Differentiated Pluripotent Stem Cell Progeny Depleted of Extraneous Phenotypes

ABSTRACT

The invention provides methods for depleting extraneous phenotypes from a mixed population of cells comprising the in vitro differentiated progeny of primate pluripotent stem cells. The invention also provides mixed cell populations enriched for a target cell phenotype where the mixed cell population comprises the differentiated in vitro progeny of primate embryonic stem cells.

PRIORITY

This application claims priority to provisional application No.61/220,418, filed Jun. 25, 2009.

FIELD OF THE INVENTION

The invention relates to the field of stem cell biology.

BACKGROUND

Pluripotent stem cells have the ability to both proliferate in cultureand, under appropriate growth conditions, differentiate into lineagerestricted cell types representative of all three primary germ layers:endoderm, mesoderm and ectoderm (U.S. Pat. Nos. 5,843,780; 6,200,806;7,029,913; Shamblott et al., (1998) Proc. Natl. Acad Sci. USA 95:13726;Takahashi et al., (2007) Cell 131(5):861; Yu et al., (2007) Science318:5858). Defining appropriate growth conditions for particular lineagerestricted cell types will provide virtually an unlimited supply of thatcell type for use in research and therapeutic applications.

Protocols for differentiating primate pluripotent stem (pPS) cells intoa variety of targeted cell types including oligodendrocytes, neuronalcells, cardiomyocytes, hematopoietic cells, pancreatic islet cells,hepatocytes, osteoblast and chondrocytes have been described (see, e.g.,U.S. Pat. Nos. 7,285,415; 6,833,269; 7,425,448; 7,452,718; 7,033,831;7,326,572; 6,458,589; 6,506,574; 7,256,042; 7,473,555; U.S. PatentPublication Nos. 2005/0158855; 2004/0224403; 2005/0282272; 2005/0282274;2006/0148077; U.S. patent application Ser. No. 12/412,183; PCTPublication No. WO 07/149182; WO 05/097980; Carpenter et al. (2001) ExpNeurology 172:383; Chadwick et al. (2003) Blood 102:906; Kierstad et al.(2005) J Neuroscience 25:4694); Laflamme et al. (2007) NatureBiotechnology 25:1015 Jiang et al. (2007) Stem Cells 25:1940.

Differentiation of pPS cells into a target phenotype cell may result inproduction of a mixed population of cells comprising the targetedphenotype as well as various extraneous phenotypes. Certain extraneousphenotypes that retain the pluripotent potential of undifferentiatedembryonic stem cells may form teratomas when administered to a subject,see, e.g., Thomson 1998 Science 282:1145. Other extraneous phenotypesmay interfere with the efficacy of the target phenotype merely bydiluting the number of cells of the targeted phenotype thereby reducingthe overall efficacy of the cell preparation. Accordingly, there is aneed to reduce the number of cells having an extraneous phenotype foundin a population of cells comprising the targeted differentiated progenyof pPS cells. There is an additional need for populations ofdifferentiated progeny of pPS cells that have a minimal number of cellsof an extraneous phenotype for use in therapeutic, diagnostic and/orresearch applications. Various embodiments of the invention describedherein meet these needs and other needs as well.

SUMMARY OF THE INVENTION

In some embodiments the invention provides a population of cellscomprising the in vitro differentiated progeny of pPS cells that isessentially free of extraneous phenotypic cell types. The in vitrodifferentiated progeny of pPS cells may include target phenotypes chosenfrom oligodendrocytes, cardiomyocytes, islet cells, hepatocytes,hematopoietic cells, chondrocytes and osteoblasts. One example ofextraneous phenotypic cells is an epithelial lineage cell, i.e. a cellexpressing one or more markers associated with epithelial cells, such asone or more of the following markers: cytokeratin, epithelial celladhesion molecule (EpCAM) desmocollin 3; desmoglein 2; E-cadherin CD49f,EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105 (endoglin). These cellsmay be capable of forming clustered epithelial structures when implantedin a subject such as a rodent. Cell populations that are essentiallyfree of extraneous phenotypic cell types may be obtained by screeningfor markers associated with epithelial cells such as one or more of thefollowing markers: desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA,E-cad, CD321 (Jam1), CD10, CD66, and CD105 (endoglin) cytokeratin. Forexample an antibody to any one or more of these markers may be used.

In other embodiments the invention provides a method of obtaining apopulation of cells comprising the in vitro differentiated progeny ofpPS cells that is essentially free of extraneous phenotypic cellscomprising a) obtaining a population of cells comprising the in vitrodifferentiated progeny of pPS cells; b) contacting the cell populationof a) with one or more ligands that bind to epithelial cells and c)removing the ligand bound cells of b) thereby obtaining a population ofcells that is essentially free of extraneous phenotypic cells. Removingthe ligand bound cell can include physically separating the ligand boundcell from the rest of cell population. Removing the ligand bound cellmay also include killing the ligand bound cell. For example an agentsuch as toxin that binds to the ligand bound to the cell may used. Inone embodiment the ligand may be antibody and complement may be used asagent that lyses the cell and thus removes it from the population ofcells.

In certain other embodiments the invention provides for a mixedpopulation of cells comprising the in vitro differentiated progeny ofprimate pluripotent stem cells. The mixed population of cells mayinclude a targeted phenotypic cell population that is enriched byreducing the number of extraneous phenotypic cells from the mixedpopulation of cells thus providing a more suitable population of cellsfor use as a therapeutic or research product when compared to a mixedpopulation of cells that has not been reduced in the number ofextraneous phenotypic cells. Enrichment of the targeted population ofcells is achieved by reducing the number of cells that express specificmarkers associated with cells of extraneous phenotype, e.g. one or moremarkers expressed by an epithelial cell. Removal of the extraneousphenotype may include physically separating the extraneous phenotypefrom other cells comprising the population. Removal of the extraneousphenotype may also include chemically treating the extraneous phenotypeto kill the ligand bound cell e.g. a chemical agent that specificallybinds to a ligand bound to an extraneous phenotypic cell.

Surprisingly, expression of at least one or more markers associated withextraneous phenotypic cells of epithelial lineage overlaps withexpression of at least one or more markers associated withundifferentiated cells, e.g. TRA-1-60. Thus by depleting cellsexpressing the markers associated with extraneous phenotypes ofepithelial lineage the number of undifferentiated cells, or the numberof cells expressing at least one marker expressed by an undifferentiatedcell (such as TRA-1-60) within the mixed population may also be reduced.

In some embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to the extraneous phenotypic cells; andb) separating the ligand bound extraneous phenotypic cells from the restof the mixed population of cells thereby reducing the number ofextraneous phenotypic cells from a mixed population of cells, whereinthe mixed population of cells comprises the in vitro differentiatedprogeny of pPS cells including the extraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to the extraneous phenotypic cells; andb) contacting the ligand bound extraneous phenotypic cells with achemical agent that kills the ligand bound extraneous phenotype cellthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In certain embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to one or more markers expressed by anepithelial cell; and b) separating the ligand bound cells of a) from therest of the mixed population of cells thereby reducing the number ofextraneous phenotypic cells from a mixed population of cells, whereinthe mixed population of cells comprises the in vitro differentiatedprogeny of pPS cells including the extraneous phenotypic cells.

In some embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to one or more markers expressed by anepithelial cell; and b) contacting the ligand bound cells of a) with achemical agent that kills the ligand bound cells of a) thereby reducingthe number of extraneous phenotypic cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind epithelial cells and contacting the mixedpopulation of cells with one or more ligands that specifically bind toundifferentiated pPS cells; and b) separating the ligand bound cellsfrom the rest of the mixed population of cells thereby reducing thenumber of extraneous phenotypic cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In still other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind epithelial cells and contacting the mixedpopulation of cells with one or more ligands that specifically bind toundifferentiated pPS cells; and b) contacting the ligand bound cells ofa) with a chemical agent that kills the ligand bound cells of a) therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In yet other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a molecule chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin); and b) separating the ligand bound cells from the rest ofthe mixed population of cells thereby reducing the number of extraneousphenotypic cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the extraneous phenotypic cells.

In yet other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a molecule chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin); and b) contacting the ligand bound cells of a) with achemical agent that kills the ligand bound cells of a) thereby reducingthe number of extraneous phenotypic cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands chosen from cytokeratin, epithelial cell adhesion molecule(EpCAM) desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321(Jam1), CD10 CD66, and CD105 (endoglin); b) contacting the mixedpopulation of cells with one or more ligands that specifically bind toundifferentiated pPS cells; and c) contacting the ligand bound cells ofa), b) or a) and b) with a with a chemical agent that kills the ligandbound cells, thereby reducing the number of extraneous phenotypic cellsfrom a mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theextraneous phenotypic cells.

In still further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands chosen from cytokeratin, epithelial cell adhesion molecule(EpCAM) desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321(Jam1), CD10, CD66, and CD105 (endoglin); b) contacting the mixedpopulation of cells with a ligand that specifically bind to TRA-160; andc) separating the ligand bound cells from the rest of the mixedpopulation of cells thereby reducing the number of extraneous phenotypiccells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the extraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands chosen from cytokeratin, epithelial cell adhesion molecule(EpCAM) desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321(Jam1), CD10, CD66, and CD105 (endoglin); b) contacting the mixedpopulation of cells with a ligand that specifically bind to TRA-160; andc) contacting the ligand bound cells of a), b) or a) and b) with a witha chemical agent that kills the ligand bound cells thereby reducing thenumber of extraneous phenotypic cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In certain other embodiments the invention provides a method of reducingthe number of epithelial cells in a mixed population of cells comprisinga) contacting the mixed population of cells with one or more ligandsthat specifically bind epithelial cells and b) separating the ligandbound epithelial cells from the rest of the mixed population of cellsthereby reducing the number of epithelial cells from a mixed populationof cells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In certain other embodiments the invention provides a method of reducingthe number of epithelial cells in a mixed population of cells comprisinga) contacting the mixed population of cells with one or more ligandsthat specifically bind epithelial cells and b) contacting the cells ofa) with a chemical agent that kills the ligand bound cells of a) therebyreducing the number of epithelial cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In further embodiments the invention provides a method of reducing thenumber of epithelial cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind one or more molecules chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin) and b) separating the ligand bound epithelial cells from therest of the mixed population of cells thereby reducing the number ofepithelial cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the extraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind one or more molecules chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin) and b) contacting the ligand bound epithelial cells with achemical agent that kills the ligand bound cells thereby reducing thenumber of epithelial cells from a mixed population of cells, wherein themixed population of cells comprises the in vitro differentiated progenyof pPS cells including the extraneous phenotypic cells.

In still other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and b) separating the EpCAMbound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and b) contacting the EpCAMbound cells with a chemical agent that kills the EpCAM bound cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a cytokeratin molecule and b)separating the cytokeratin bound cells from the rest of the mixedpopulation of cells thereby reducing the number of extraneous phenotypiccells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the extraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a cytokeratin molecule and b)contacting the cytokeratin bound cells with a chemical agent that killsthe ligand bound cells thereby reducing the number of extraneousphenotypic cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the extraneous phenotypic cells.

In some embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a desmocollin 3 molecule and b)separating the desmocollin 3 bound cells from the rest of the mixedpopulation of cells thereby reducing the number of extraneous phenotypiccells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the extraneous phenotypic cells.

In some embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a desmocollin 3 molecule and b)contacting the desmocollin 3 bound cells with a chemical agent thatkills the ligand bound cells thereby reducing the number of extraneousphenotypic cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the extraneous phenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a desmoglein 2 molecule and b)separating the desmoglein 2 bound cells from the rest of the mixedpopulation of cells thereby reducing the number of extraneous phenotypiccells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the extraneous phenotypic cells.

In still further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a desmoglein 2 molecule and b)contacting the desmoglein 2 bound cells with a chemical agent that killsthe ligand bound cells thereby reducing the number of extraneousphenotypic cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the extraneous phenotypic cells.

In still further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a E-cadherin molecule and b)separating the E-cadherin bound cells from the rest of the mixedpopulation of cells thereby reducing the number of extraneous phenotypiccells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the extraneous phenotypic cells.

In yet further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a E-cadherin molecule and b)contacting the E-cadherin bound cells with a chemical agent that killsthe ligand bound cells thereby reducing the number of extraneousphenotypic cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the extraneous phenotypic cells.

In still other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD49f molecule and b) separating theCD49f bound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD49f molecule and b) contacting theCD49f bound cells with a chemical agent that kills the ligand boundcells thereby reducing the number of extraneous phenotypic cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theextraneous phenotypic cells.

In still other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a EMA molecule and b) separating theEMA bound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In some embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a EMA molecule and b) contacting theEMA bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to an E-cad molecule and b) separatingthe E-cad bound cells from the rest of the mixed population of cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In yet further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to an E-cad molecule and b) contactingthe E-cad bound cells with a chemical agent that kills the ligand boundcells thereby reducing the number of extraneous phenotypic cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theextraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD321 molecule and b) separating theCD321 bound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In still other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD321 molecule and b) contacting theCD321 bound cells with a chemical agent that kills the ligand boundcells f thereby reducing the number of extraneous phenotypic cells froma mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theextraneous phenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD66 molecule and b) separating theCD66 bound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In yet further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD66 molecule and b) contacting theCD66 bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD10 molecule and b) separating theCD10 bound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD10 molecule and b) contacting theCD10 bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In still further embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD105 molecule and b) separating theCD105 bound cells from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to a CD105 molecule and b) contacting theCD105 bound cells with a chemical agent that kills the ligand boundcells thereby reducing the number of extraneous phenotypic cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theextraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of cytokeratin expressing cells from a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and b) separating the EpCAMbound cells from the rest of the mixed population of cells therebyreducing the number of cytokeratin cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the cytokeratin expressingcells.

In certain other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and one or more ligands thatspecifically bind to a marker expressed on an undifferentiated cell; andb) separating the cells bound to the ligand for EpCAM and the cellsbound to the ligand of a marker expressed on undifferentiated cells fromthe rest of the mixed population of cells thereby reducing the number ofextraneous phenotypic cells from a mixed population of cells, whereinthe mixed population of cells comprises the in vitro differentiatedprogeny of pPS cells including the extraneous phenotypic cells.

In other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and one or more ligands thatspecifically bind to a marker expressed on an undifferentiated cell; andb) contacting the cells bound to the ligand for EpCAM and/or the cellsbound to the ligand of a marker expressed on undifferentiated cells witha chemical agent that kills the ligand bound cells thereby reducing thenumber of extraneous phenotypic cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In yet other embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and one or more ligands thatspecifically bind to TRA-1-60; and b) separating the cells bound to theligand for EpCAM and the cells bound to the ligand of TRA-1-60 from therest of the mixed population of cells thereby reducing the number ofextraneous phenotypic cells from a mixed population of cells, whereinthe mixed population of cells comprises the in vitro differentiatedprogeny of pPS cells including the extraneous phenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to EpCAM and one or more ligands thatspecifically bind to TRA-1-60; and b) contacting the cells bound to theligand for EpCAM and/or the cells bound to the ligand of TRA-1-60 with achemical agent that kills the ligand bound cells thereby reducing thenumber of extraneous phenotypic cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In certain other embodiments the invention provides a method of reducingthe number of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to one or more molecules chosen fromcytokeratin, epithelial cell adhesion molecule (EpCAM) desmocollin 3;desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66,and CD105 (endoglin) and one or more ligands that specifically bind to amarker expressed on an undifferentiated cell; and b) separating thecells bound to the one or more molecules chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin) and the cells bound to the ligand of a marker expressed onundifferentiated cells from the rest of the mixed population of cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.

In further embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to one or more molecules chosen fromcytokeratin, epithelial cell adhesion molecule (EpCAM) desmocollin 3;desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66,and CD105 (endoglin) and one or more ligands that specifically bind to amarker expressed on an undifferentiated cell; and b) contacting thecells bound to the one or more ligands specifically to bound to one ormore molecules chosen from cytokeratin, epithelial cell adhesionmolecule (EpCAM) desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA,E-cad, CD321 (Jam1), CD10, CD66, and CD105 (endoglin) and/or the cellsbound to the ligand of a marker expressed on undifferentiated cells witha chemical agent that kills the ligand bound cells thereby reducing thenumber of extraneous phenotypic cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the extraneous phenotypiccells.

In yet other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to one or more molecules chosen fromcytokeratin, epithelial cell adhesion molecule (EpCAM) desmocollin 3;desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66,and CD105 (endoglin) and b) separating the ligand bound cells from therest of the mixed population of cells thereby reducing the number ofepithelial cluster forming cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the epithelial clusterforming cells.

In further embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to one or more molecules chosen fromcytokeratin, epithelial cell adhesion molecule (EpCAM) desmocollin 3;desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66,and CD105 (endoglin) and b) contacting the ligand bound cells of a) witha chemical agent that kills the ligand bound cells thereby reducing thenumber of epithelial cluster forming cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells including the epithelial clusterforming cells.

In yet other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to EpCAM and b) separating the EpCAMbound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to EpCAM and b) contacting the EpCAMbound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to a cytokeratin and b) separatingthe cytokeratin bound cells from the rest of the mixed population ofcells thereby reducing the number of epithelial cluster forming cellsfrom a mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In further embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to a cytokeratin and b) contactingthe cytokeratin bound cells with a chemical agent that kills the ligandbound cells thereby reducing the number of epithelial cluster formingcells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the epithelial cluster forming cells.

In yet further embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to desmocollin 3 and b) separatingthe desmocollin 3 bound cells from the rest of the mixed population ofcells thereby reducing the number of epithelial cluster forming cellsfrom a mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to desmocollin 3 and b) contactingthe desmocollin 3 bound cells with a chemical agent that kills theligand bound cells thereby reducing the number of epithelial clusterforming cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells including the epithelial cluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to desmoglein 2 and b) separatingthe desmoglein 2 bound cells from the rest of the mixed population ofcells thereby reducing the number of epithelial cluster forming cellsfrom a mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to desmoglein 2 and b) contactingthe desmoglein 2 bound cells with a chemical agent that kills the ligandbound cells thereby reducing the number of epithelial cluster formingcells from a mixed population of cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cellsincluding the epithelial cluster forming cells.

In still further embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to E-cadherin and b) separating theE-cadherin bound cells from the rest of the mixed population of cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In further embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to E-cadherin and b) contacting theE-cadherin bound cells with a chemical agent that kills the ligand boundcells thereby reducing the number of epithelial cluster forming cellsfrom a mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD49f and b) separating the CD49fbound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD49f and b) contacting the CD49fbound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to EMA and b) separating the EMAbound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to EMA and b) contacting the EMAbound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In further embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to a E-cad and b) separating theE-cad bound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In still further embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to a E-cad and b) contacting theE-cad bound cells with a chemical agent that kills the ligand boundcells thereby reducing the number of epithelial cluster forming cellsfrom a mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In yet other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD321 and b) separating the CD321bound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In yet other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD321 and b) contacting the CD321bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD66 and b) separating the CD66bound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD66 and b) contacting the CD66bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD10 and b) separating the CD10bound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD10 and b) contacting the CD10bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells

In still other embodiments the invention provides a method of reducingthe number of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD105 and b) separating the CD105bound cells from the rest of the mixed population of cells therebyreducing the number of epithelial cluster forming cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the epithelialcluster forming cells.

In other embodiments the invention provides a method of reducing thenumber of epithelial cluster forming cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to CD105 and b) contacting the CD105bound cells with a chemical agent that kills the ligand bound cellsthereby reducing the number of epithelial cluster forming cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theepithelial cluster forming cells.

In further embodiments the invention provides a method of reducing thenumber of undifferentiated cells in a mixed population of cellscomprising a) contacting the mixed population of cells with one or moreligands that specifically bind to the undifferentiated cells; and b)removing the ligand bound undifferentiated cells from the rest of themixed population of cells thereby reducing the number ofundifferentiated cells from a mixed population of cells wherein themixed population of cells comprises the in vitro differentiated progenyof pPS cells. In some embodiments the ligand may include an antibody,such as an IgG that binds specifically to TRA-1-60.

In yet other embodiments the invention provides a method of reducing thenumber of cells expressing one or more molecules expressed byundifferentiated cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind to an epithelial cell; and b) removing the ligandbound cells of a) from the rest of the mixed population of cells therebyreducing the number of undifferentiated cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.

In further embodiments the invention provides a method of reducing thenumber of cells expressing one or more molecules expressed byundifferentiated cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind to EpCAM and b) removing the ligand bound EpCAM cellsfrom the rest of the mixed population of cells thereby reducing thenumber of undifferentiated cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.

In still other embodiments the invention provides a method of reducingthe number of cells expressing one or more molecules expressed byundifferentiated cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind to EpCAM and one or more ligands that specificallybind to TRA-1-60 and b) removing the ligand bound cells from the rest ofthe mixed population of cells thereby reducing the number ofundifferentiated cells from a mixed population of cells, wherein themixed population of cells comprises the in vitro differentiated progenyof pPS cells.

In yet other embodiments the invention provides a method of reducing thenumber of cells expressing one or more molecules expressed byundifferentiated cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind to a cytokeratin and b) removing the ligand boundcytokeratin cells from the rest of the mixed population of cells therebyreducing the number of undifferentiated cells from a mixed population ofcells, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.

In further embodiments the invention provides a method of reducing thenumber of cells expressing one or more molecules expressed byundifferentiated cells in a mixed population of cells comprising a)contacting the mixed population of cells with one or more ligands thatspecifically bind to a cytokeratin and one or more ligands thatspecifically bind to TRA-1-60 and b) removing the ligand bound cellsfrom the rest of the mixed population of cells thereby reducing thenumber of undifferentiated cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.

In yet other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing a molecule found on an epithelial cell.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing a molecule found on an epithelial cell and atleast one cell expressing a molecule found on an undifferentiated stemcell.

In other embodiments the invention provides a mixed population of cellsthat is enriched for a targeted phenotype, wherein the mixed populationof cells comprises the in vitro differentiated progeny of pPS cells andwherein the mixed population of cells has been depleted of at least onecell expressing one or more of the molecules chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin).

In yet further embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing EpCAM.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing a cytokeratin.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing desmocollin 3.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing desmoglein 2.

In other embodiments the invention provides a mixed population of cellsthat is enriched for a targeted phenotype, wherein the mixed populationof cells comprises the in vitro differentiated progeny of pPS cells andwherein the mixed population of cells has been depleted of at least onecell expressing E-cadherin.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing CD49f.

In further embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing EMA.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing E-cad.

In yet other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing CD321.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing CD166.

In still other embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing CD105.

In further embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing one or more molecules chosen from cytokeratin,epithelial cell adhesion molecule (EpCAM) desmocollin 3; desmoglein 2;E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105(endoglin) and at least one cell expressing a marker found on anundifferentiated primate pluripotent cell

In other embodiments the invention provides a mixed population of cellsthat is enriched for a targeted phenotype, wherein the mixed populationof cells comprises the in vitro differentiated progeny of pPS cells andwherein the mixed population of cells has been depleted of at least onecell expressing EpCAM and at least one cell expressing a marker found onan undifferentiated primate pluripotent cell.

In still further embodiments the invention provides a mixed populationof cells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing a cytokeratin and at least one cell expressinga marker found on an undifferentiated primate pluripotent stem cell.

In other embodiments the invention provides a mixed population of cellsthat is enriched for a targeted phenotype, wherein the mixed populationof cells comprises the in vitro differentiated progeny of pPS cells andwherein the mixed population of cells has been depleted of at least onecell expressing EpCAM and at least one cell expressing TRA-1-60.

In yet further embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells and wherein the mixed population of cells has been depleted of atleast one cell expressing a cytokeratin and at least one cell expressingTRA-1-60.

In still other embodiments the invention provides a method oftransplanting into a subject a population of cells comprising cellshaving a targeted phenotype comprising a) obtaining a population ofcells comprising the targeted phenotype, wherein the population of cellshas been depleted of a least one cell having an extraneous phenotype;and b) administering the population of cells from a) to the subject.

In yet other embodiments the invention provides a method of treating asubject in need of cellular therapy comprising a) obtaining a populationof cells comprising cells having a targeted phenotype, wherein thepopulation of cells has been depleted of a least one cell having anextraneous phenotype and b) administering the population of cells froma) to the subject.

In further embodiments the invention provides the use of a population ofcells for treating a subject in need of cellular therapy comprisingcells having a targeted phenotype, wherein the population of cells hasbeen depleted of a least one cell having an extraneous phenotype.

In still other embodiments the invention provides a method oftransplanting into a subject a population of cells comprising cellshaving a targeted phenotype comprising a) obtaining a population ofcells comprising cells having a targeted phenotype, wherein thepopulation of cells has been depleted of a least one cell expressing anepithelial cell marker; and b) administering the population of cellsfrom a) to the subject.

In yet other embodiments the invention provides a method of treating asubject in need of cellular therapy comprising a) obtaining a populationof cells comprising cells having a targeted phenotype, wherein thepopulation of cells has been depleted of a least one cell expressing anepithelial cell marker; and b) administering the population of cellsfrom a) to the subject.

In further embodiments the invention provides the use of a population ofcells for treating a subject in need of cellular therapy comprisingcells having a targeted phenotype, wherein the population of cells hasbeen depleted of a least one cell expressing an epithelial cell marker.

In other embodiments the invention provides a kit for depleting cellshaving an extraneous phenotype from a mixed population of cellscomprising a) a ligand for one or more molecules expressed on anepithelial cell; b) a ligand for one or more molecules expressed on anundifferentiated cell; and c) one or more containers, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells.

In other embodiments the invention provides a kit for depleting cellshaving an extraneous phenotype from a mixed population of cellscomprising a) a ligand for one or more molecules chosen fromcytokeratin, epithelial cell adhesion molecule (EpCAM) desmocollin 3;desmoglein 2; E-cadherin CD49f, epithelial membrane antigen (EMA),E-cad, CD321 (Jam1), CD10, CD66, and CD105 (endoglin); b) a ligand forone or more molecules expressed on an undifferentiated cell; and c) oneor more containers, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells.

In further embodiments the invention provides a kit for depleting cellshaving an extraneous phenotype from a mixed population of cellscomprising a ligand for EpCAM, a ligand for TRA-1-60 and one or morecontainers, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.

In further embodiments the invention provides a kit for depleting cellshaving an extraneous phenotype from a mixed population of cellscomprising a ligand for a cytokeratin, a ligand for TRA-1-60 and one ormore containers, wherein the mixed population of cells comprises the invitro differentiated progeny of pPS cells.

In other embodiments the invention provides a kit for depleting cellshaving an extraneous phenotype from a mixed population of cellscomprising a) a ligand for one or more molecules chosen fromcytokeratin, epithelial cell adhesion molecule (EpCAM) desmocollin 3;desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66,and CD105 (endoglin); and b) one or more containers, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells.

In still other embodiments the invention provides an antibody that bindsspecifically to TRA-1-60 wherein the antibody has an IgG isotype.

DESCRIPTION OF THE FIGURES

FIG. 1 is a photomicrograph showing the clustered epithelial structures,present in a small percentage of rats administered oligodendroglialprogenitor cells differentiated in vitro from hES cells, have anepithelial-like morphology.

FIG. 2 is a series of photomicrographs showing that the pan-cytokeratinantibody AE1/AE3 labels a variety of tissue types found in a humantissue array (panels on left side) and also labels murine teratomaformed by injecting the mice intra-muscularly with undifferentiated hEScells (panels on right side).

FIG. 3 are photomicrographs showing that the Ber-EP4 (EpCAM) antibodylabels a variety of tissue types found in a human tissue array (panelson left side) and also labels murine teratomas formed by injecting themice intra-muscularly with undifferentiated hES cells (panels on rightside).

FIG. 4 is a series of photomicrographs showing clustered epithelialstructures obtained from 3 rats administered a preparation ofoligodendroglial progenitor cells differentiated in vitro from hES cellslabeled positively in some cases with the pan-cytokeratin antibodyAE1/AE3. Upper panel magnification is 24×. Lower panel magnification is400×.

FIG. 5 is a series of photomicrographs showing clustered epithelialstructures obtained from one rat administered a preparation ofoligodendroglial progenitor cells differentiated in vitro from hES cellslabeled in some cases with the Ber-EP4 EpCAM antibody.

FIG. 6 is a series of photomicrographs showing clustered epithelialstructures obtained from one rat administered a preparation ofoligodendroglial progenitor cells differentiated in vitro from hES cellslabeled in some cases with both the Ber-EP4 EpCAM antibody and thepan-cytokeratin antibody AE1/AE3.

FIG. 7 is a series of photomicrographs showing clustered epithelialstructures obtained from one rat administered a preparation ofoligodendroglial progenitor cells differentiated in vitro from hES cellslabeled in some cases with the pan-cytokeratin antibody AE1/AE3, but notthe Ber-EP4 (EpCAM) antibody.

FIG. 8 is a series of photomicrographs showing clustered epithelialstructures obtained from one rat administered a preparation ofoligodendroglial progenitor cells differentiated in vitro from hES cellsin which cells were labeled with neither the pan-cytokeratin antibodyAE1/AE3, or the Ber-EP4 (EpCAM) antibody.

FIG. 9 shows data acquired by flow cytometry. Two different lots ofoligodendroglial progenitor cells differentiated in vitro from humanembryonic stern (hES) cells were labeled for EpCam, pan-cytokeratin andTRA-1-60.

FIG. 10A and FIG. 10B show data acquired by flow cytometry. Cells thatdo not express EpCAM (HEK 293) were spiked with EpCAM+ cells to create amixed population. The mixed population was then selected to separationusing magnetic beads conjugated to an EpCAM-specific antibody. Theresulting fractions were analyzed by flow cytometry using either anEpCAM specific antibody followed by labeled secondary antibody (A647) oran isotype control followed by the same secondary antibody. FIG. 10Cshows that efficiency of recovery of the EpCAM negative cells wascalculated to be 93% and efficiency of recovery of the EpCAM positivecells was calculated to be 9%.

FIG. 11 shows data acquired by flow cytometry. A single lot ofoligodendroglial progenitor cells differentiated in vitro from hES cellswas depleted of EpCAM positive cells using an EpCAM-specific antibodyconjugated to a magnetic bead. The resulting cell fractions were labeledfor TRA-1-60 and pan-cytokeratin using antibodies specific for each. TheTRA-1-60 labeled cells were stained with a secondary antibody as well.

DEFINITIONS

“About,” as used herein to refer to an amount or a value means+ or −5%of stated amount or value.

“Cardiomyocytes,” as used herein includes mature cardiomyocytes (a cellthat possesses the functional capability of a cardiomyocyte isolatedfrom either a juvenile or adult primate) and/or cardiomyocyte precursors(cells which express one or markers expressed on, in, or bycardiomyocytes and/or have one or more morphological attributesassociated with cardiomyocytes and which may differentiate into maturecardiomyocyte either in vitro or when implanted into a subject).

“Cell culture,” as used herein, refers to a plurality of cells grown invitro over time. The cell culture may originate from a plurality of pPScells or from a single pPS cell and may include all of the progeny ofthe originating cell or cells, regardless of 1) the number of passagesor divisions the cell culture undergoes over the lifetime of theculture; and 2) any changes in phenotype to one or more cells within theculture over the lifetime of the culture. Thus, as used herein, a cellculture begins with the initial seeding of one or more suitable vesselswith at least one pPS cell and ends when the last surviving progeny ofthe original founder(s) is harvested or dies. Seeding of one or moreadditional culture vessels with progeny of the original founder cells isalso considered to be a part of the original cell culture.

“Chondrocyte,” as used herein, includes mature chondrocytes (a cell thatpossesses the functional capability of a chondrocyte isolated fromeither a juvenile or adult primate) and/or chondrocyte precursors (cellswhich express one or markers expressed on, in, or by chondrocytes and/orhave one or more morphological attributes associated with chondrocytesand which may differentiate into mature chondrocytes either in vitro orwhen implanted into a subject).

“Clustered epithelial structures” as used herein, refers to a structurethat may form when extraneous phenotypic cells of epithelial lineagefound in a mixed population of cells comprising the in vitrodifferentiated progeny of pPS cells are implanted into a subject. Thesestructures have the morphology of epithelial cells and express at leastone molecular marker expressed in epithelial cells as described infra.In some instances the structures may have the form of a sac or vesicle.

“Cytokeratin,” as used herein, refers to intermediate filament keratinsfound in the intracytoplasmic cytoskeleton of epithelial tissue. Thereare two types of cytokeratins: acidic types I cytokeratins and basic orneutral type II cytokeratins. Cytokeratins are usually found in pairscomprising a type I cytokeratin and a type II cytokeratin. The type IIcytokeratins, which are the basic or neutral cytokeratins, comprisesubtypes CK1, CK2, CK3, CK4, CK5, CK6a, CK6b, CK6c, CK7 and CK80. Thetype I cytokeratins, which are the acidic cytokeratins, comprisesubtypes CK9, CK10, CK12, CK13, CK14, CK15, CK16, CK17, CK18, CK19,CK20, CK23 and CK24. All cytokeratin proteins contain a centralα-helix-rich domain (with a 50-90% sequence identity among cytokeratinsof the same type and around 30% between cytokeratins of different types)with non-α-helical N- and C-terminal domains.

“Depleted,” as used herein, refers to an act by which an extraneousphenotype contained within a mixed population of cells has beendecreased in number relative to other phenotypic cell types within thepopulation as a result of an act initiated by the human hand. Includedare methods where the extraneous phenotype is physically segregated fromthe other cells in the population, as for example byimmuno-precipitating the extraneous phenotypic cells. Also included aremethods where the extraneous phenotypic cells are removed chemicallyfrom the rest of the population, e.g., where the extraneous phenotypiccell is specifically targeted with a chemical agent such as a toxin,complement and the like. Thus cell populations depleted of one or morecells having an extraneous phenotype are considered to be depleted.Typically the depletion is measured by comparing the number of cellsbearing the extraneous phenotype before and after the act initiated bythe human hand whereby any decrease in the relative number of the cellpopulation bearing the extraneous phenotype as a result of the actinitiated by the human hand is considered depletion. Depletion of anextraneous phenotype from a mixed population of cells may result in theenrichment of a target cell population within the mixed population ofcells.

“Desmocollin 3,” as used herein, refers to an epithelial cell markerthat is a calcium-dependent glycoprotein that is a member of thedesmocollin subfamily of the cadherin superfamily. These desmosomalfamily members, along with the desmogleins, are found primarily inepithelial cells where they constitute the adhesive proteins of thedesmosome cell-cell junction and are required for cell adhesion anddesmosome formation. The desmosomal family members are arranged in twoclusters on chromosome 18, occupying less than 650 kb combined.Alternative splicing results in two transcript variants encodingdistinct isoforms.

“Desmoglein 2,” as used herein, refers to an epithelial cell marker.Desmoglein 2 is a calcium-binding transmembrane glycoprotein componentof desmosomes in vertebrate epithelial cells. Desmosomes are cell-celljunctions between epithelial, myocardial, and certain other cell types.Currently, three desmoglein subfamily members have been identified andall are members of the cadherin cell adhesion molecule superfamily.These desmoglein gene family members are located in a cluster onchromosome 18. This second family member is expressed in colon, coloncarcinoma, and other simple and stratified epithelial-derived cells.

“E-cadherin” (CD324), as used herein, refers to an epithelial cellmarker expressed on the cell surface. It is a calcium dependentcell-cell adhesion glycoprotein comprised of five extracellular cadherinrepeats, a transmembrane region and a highly conserved cytoplasmic tail.Mutations in this gene are correlated with gastric, breast, colorectal,thyroid and ovarian cancer. Loss of function is thought to contribute toprogression in cancer by increasing proliferation, invasion, and/ormetastasis. The ectodomain of this protein mediates bacterial adhesionto mammalian cells and the cytoplasmic domain is required forinternalization.

“Enriched,” as use herein, refers to an act initiated by the human handby which a target phenotype contained within a mixed population of cellshas increased in number relative to other phenotypic cell types withinthe population. Typically the enrichment is measured by comparing thenumber of target phenotypic cells before and after the act initiated bythe human hand whereby any increase in the relative number of thetargeted cell population as a result of the act initiated by the humanhand is considered enrichment.

“Embryoid bodies,” as used herein, refers to heterogeneous clusterscomprising undifferentiated, differentiated and partly differentiatedcells that appear when primate pluripotent stem cells are allowed todifferentiate in a non-specific fashion in suspension cultures oraggregates.

As used herein, “embryonic stem cell” (ES) refers to pluripotent stemcells that are 1) derived from a blastocyst before substantialdifferentiation of the cells into the three germ layers or 2)alternatively obtained from an established cell line. Except whereexplicitly required otherwise, the term includes primary tissue andestablished lines that bear phenotypic characteristics of ES cells, andprogeny of such lines that have the pluripotent phenotype. The ES cellsmay be human ES cells (hES). Prototype “human Embryonic Stem cells” (hEScells) are described by Thomson et al. (Science 282:1145, 1998; U.S.Pat. No. 6,200,806) and may be obtained from any one of a number ofestablished stem cell banks such as the UK Stem Cell Bank(Hertfordshire, England) and the National Stem Cell Bank (Madison,Wis.).

“EpCAM,” as used herein, refers to Epithelial Cell Adhesion Molecule anepithelial cell marker. It is a pan-epithelial differentiation antigenthat is expressed on epithelial cells and almost all carcinomas.

“Epithelial cells,” as used herein, refers to cells having a morphologychosen from a) squamous (thin flat); b) cuboidal (cube like); c)columnar (tall) d) pseudostratified (all cells reach the basementmembrane, and some extend all the way to the surface, while others donot; e) transitional (e.g. as found in the urinary bladder) where cellsmay appear squamous under some conditions and rounded under otherconditions; and express at least one marker associated with epithelialcells. Exemplary epithelial markers include EpCAM; cytokeratin;desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1),CD10, CD66, and CD105 (endoglin).

“Extraneous phenotype,” as used herein, refers to one or more cell typescontained within a mixed population of cells that are undesirable. Thusin a mixed population of cells comprising a target phenotypic cellpopulation, any cell having a phenotype that differs from the targetpopulation may be considered extraneous. Extraneous phenotypic cells maybe targeted for depletion. An example of an extraneous phenotypic cellis a cell that has the potential to form a clustered epithelialstructure when administered to a subject and that expresses one or moremarkers expressed by an epithelial cell.

“Hematopoletic cells,” as used herein, includes mature hematopoieticcells (a cell that possesses the functional capability of ahematopoietic cell isolated from either a juvenile or adult primate)such as dendritic cells, macrophages, B lymphocytes, T lymphocytesincluding both CD8+ and CD4+ lymphocytes, neutrophils, basophils,eosinophils, natural killer (NK) cells, platelets, erythrocytes and mastcells and/or hematopoietic precursors (cells which express one ormarkers expressed on, in, or by a hematopoietic cell and/or have one ormore morphological attributes associated with a hematopoietic cell andwhich may differentiate into mature hematopoietic cells either in vitroor when implanted into a subject).

“Hepatocytes,” as used herein, includes mature hepatocytes (a cell thatpossesses the functional capability of a hepatocyte isolated from eithera juvenile or adult primate) and/or hepatocyte precursors (cells whichexpress one or markers expressed on, in, or by hepatocytes and/or haveone or more morphological attributes associated with hepatocytes andwhich may differentiate into mature hepatocytes either in vitro or whenimplanted into a subject).

“Islet cells,” or “pancreatic islet cells,” as used herein, includesmature islet cells (a cell that possesses the functional capability ofan islet cell isolated from either a juvenile or adult primate) and/orislet cell precursors (cells which express one or more markers expressedon, in, or by islet cells and/or have one or more morphologicalattributes associated with islet cells and which may differentiate intomature islet cells either in vitro or in vivo, i.e., when implanted intoa subject).

In vitro differentiated progeny, as used herein, refers to cells thathave been differentiated from pPS cells in vitro by the addition of oneor more growth factors, cytokines, morphogens, or the like, that areadded by the human hand to a culture of pPS cells or to a culture of pPScells that have already begun to differentiate down one or more specificpathways. Additional suitable agents that may be useful indifferentiating pPS include micro RNA molecules, siRNA molecules, andsmall molecules such as IDE1, IDE2, -(−)Indolactan and Stauplimide.(see, Borowick et al. (2009) Cell Stem Cell 4:348; Chen et al. (2009)Nature Chem Biol 4:258; Zhu (2009) Cell Stem Cell 4:416). Excluded fromthe definition are the random or spontaneous differentiation eventsobtained by merely forming an embryoid body or by merely allowing aculture of pPS cells to overgrow the vessel containing them without theaddition of one or more growth factors, cytokines, morphogen or the like(beyond what may be found in commercially available cell culture mediaor commercially available serum products or serum replacement products).However, cells that have been differentiated from embryoid bodies areincluded in the definition provided that one or more growth factors,cytokines, morphogens or the like have been added to the culture tofacilitate the differentiation down a particular pathway to obtain atarget phenotype.

“Ligand,” as used herein, refers to a first molecule that specificallyinteracts with a second molecule to form a chemical bond. Typically, thebond will be a non-covalent bond such as an ionic interaction, ahydrogen bond or an interaction mediated by Van der Waals forces.Examples of ligand pairs (i.e. the first and second molecule referred toabove) include an epitope and an antibody that binds specifically tothat epitope and a substrate that specifically binds an enzyme. Thedissociation constant (K_(d)) between the two molecules is one measureof the affinity between a ligand and its specific binding partner.Specific interactions are typically characterized by relatively smallK_(d). An example of a ligand pair that bind each other with very highaffinity (very small K_(d)) is biotin/avidin. In contrast, non-specificbinding between two molecules may involve the formation of chemicalbonds based on charge and hydrophobicity, however, unlike specificbinding, the interactions are not based on the exact structure of thetwo molecules.

“Mixed population of cells,” as used herein, refers to an in vitroculture of cells comprised of more than one phenotype.

“Oligodendrocytes,” as used herein, includes mature oligodendrocytes (acell that possesses the functional capability of an oligodendrocyteisolated from either a juvenile or adult primate) and/or oligodendrocyteprecursors (cells which express one or markers expressed on, in, or byoligodendrocytes and/or have one or more morphological attributesassociated with oligodendrocytes and which may differentiate into matureoligodendrocytes either in vitro or when implanted into a subject).

“Osteoblast,” as used herein, includes mature osteoblasts (a cell thatpossesses the functional capability of an osteoblast isolated fromeither a juvenile or adult primate) and/or osteoblast precursors (cellswhich express one or markers expressed on, in, or by osteoblast and/orhave one or more morphological attributes associated witholigodendrocytes and which may differentiate into matureoligodendrocytes either in vitro or when implanted into a subject).

“Pan-cytokeratin,” as used herein, refers to an antibody or a mixture ofantibodies that bind to epitopes expressed on or in at least a pluralityof cytokeratin family members.

As used herein, “primate pluripotent stem cells” (pPS) refers to cellsthat may be derived from any source and that are capable, underappropriate conditions, of producing primate progeny of different celltypes that are derivatives of all of the 3 germinal layers (endoderm,mesoderm, and ectoderm). pPS cells may have the ability to form ateratoma in 8-12 week old SCID mice and/or the ability to formidentifiable cells of all three germ layers in tissue culture. Includedin the definition of primate pluripotent stem cells are embryonic cellsof various types including human embryonic stem (hES) cells, (see, e.g.,Thomson et al. (1998) Science 282:1145) and human embryonic germ (hEG)cells (see, e.g., Shamblott et al., (1998) Proc. Natl. Acad Sci. USA95:13726); embryonic stem cells from other primates, such as Rhesus stemcells (see, e.g., Thomson et al., (1995) Proc. Natl. Acad. Sci. USA92:7844), marmoset stem cells (see, e.g., (1996) Thomson et al., Biol.Reprod. 55:254), stem cells created by nuclear transfer technology (U.S.Patent Application Publication No. 2002/0046410), as well as inducedpluripotent stem cells (see, e.g. Yu et al., (2007) Science 318:5858);Takahashi et al., (2007) Cell 131(5):861). The pPS cells may beestablished as cell lines, thus providing a continual source of pPScells. It is contemplated that any of the embodiments of the inventiondescribed herein may be practiced by substituting one or more of thefollowing sub-groupings of pPS cells for pPS cells: human embryonic stemcells, human embryonic germ cells, rhesus stem cells, marmoset stemcells, nuclear transfer stem cells and/or induced pluripotent stemcells.

“Target phenotype” or “targeted phenotype,” as used herein, refers to adesirable cell type differentiated in vitro from a pPS cell.

As used herein, “undifferentiated primate pluripotent stem cells” refersto a cell culture where a substantial proportion of primate pluripotentstem cells and their derivatives in the population display morphologicalcharacteristics of undifferentiated cells and maintain the capacity togive rise to at least one cell type from each of the three germ layers:endoderm, mesoderm and ectoderm. It is understood that colonies ofundifferentiated cells within the population may be surrounded byneighboring cells that are partly differentiated.

Treat, treatment, treating, as used herein, means any of the following:the reduction in severity of a disease or condition; the reduction inthe duration of a disease course; the amelioration of one or moresymptoms associated with a disease or condition; the provision ofbeneficial effects to a subject with a disease or condition, withoutnecessarily curing the disease or condition, the prophylaxis of one ormore symptoms associated with a disease or condition.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates, at least in part, to the discovery thatextraneous phenotypes may sometimes exist in cell cultures comprisingthe in vitro differentiated progeny of pPS cells. Typically theseextraneous phenotypes are rare and present at low levels. The extraneousphenotypes may manifest themselves, e.g. by multiplying in vivo when thein vitro differentiated progeny of pPS cells are administered to asubject. One example of an extraneous phenotypic cell is a cell that hasthe capacity to develop into a clustered epithelial structure whenimplanted into a subject. Cells which have the capacity to develop intoa clustered epithelial structure when implanted into a subject maytypically express one or more markers expressed by an epithelial cell.

It has been discovered that certain extraneous phenotypic cells may beidentified by the presence of epithelial cell markers such as EpCAM,desmocollin 3; desmoglein 2; E-cadherin CD49f, EMA, E-cad, CD321 (Jam1),CD10, CD66, CD105 (endoglin), cytokeratin and the like. Epithelialmarkers expressed on the cell surface of the extraneous phenotypic cellsprovide suitable markers both for identifying the presence of extraneousphenotypic cells and for targeting these cells for depletion. EpCAM isan example of such a marker. EpCam may be targeted for depletion eitheralone or in combination with one of more markers described infra. ThusEpCAM may be targeted for depletion along with a marker found onundifferentiated cells such as TRA-160. Other examples include one ormore of the following markers: desmocollin 3; desmoglein 2; E-cadherinCD49f, EMA, E-cad, CD321 (Jam1), CD10, CD66, and CD105 (endoglin).Accordingly, the markers described infra may be used in targeting thesecells for removal/depletion from a mixed population of cells thatincludes cells having an extraneous phenotype or that includes cellsthat may develop into cells having an extraneous phenotype, e.g., whenimplanted into a subject.

Surprisingly, it was also discovered that certain sub-populations ofcells that express these epithelial markers also express markers foundon undifferentiated pPS cells such as TRA-1-60. Thus targeting cellsexpressing epithelial cell markers with a suitable ligand may not onlydecrease the number of extraneous phenotypic cells in the population; itmay also facilitate minimizing the number of cells expressing markersassociated with undifferentiated pPS cells in the cell culture, whichmay include pluripotent stem cells.

Methods of Reducing Extraneous Phenotypic Cells from a Mixed Populationof Cells

In certain embodiments the invention provides a method of reducing thenumber of extraneous phenotypic cells from a mixed population of cellscomprising contacting the mixed population of cells with one or moreligands that bind to the cells having the extraneous phenotype and thenseparating the extraneous phenotypic cells from the population. Themixed population of cells may comprise the in vitro differentiatedprogeny of pPS cells.

The mixed cell population may be contacted with ligand in a suitablebuffer. Suitable buffers may include phosphate buffered saline (PBS) orany commercially available cell culture media that facilitates survivalof the mixed population of cells. Typically the buffer will be anisotonic buffer and have a pH ranging from about 6.8 to about 7.7, fromabout 6.9 to about 7.6; from about 7.0 to about 7.5. In some embodimentsthe pH of the buffer will be physiological pH.

The method is adaptable over a wide range of sample volumes ranging fromsample sizes obtainable from a single well of a microwell plate to asample obtained from a scale-up bioreactor. A suitable volume forcontacting the mixed cell population can range from about 50 μL to about10,000 liters. In some embodiments a suitable volume for contacting themixture of cells is about 100 μL, about 200 μL, about 500 μL, about 1mL, about 10 mL, about 50 mL, about 100 mL, about 1 liter, about 10liters, about 50 liters, about 100 liters, about 1000 liters, about 2000liters, about 5000 liters, about 8,000 liters, about 10,000 liters ormore.

Any suitable ratio of ligands to cell number comprising the mixed cellpopulation may be used. For example a suitable ratio of ligand to cellnumber may be about 1:1; about 10:1; about 100:1; about 1000:1; about10,000:1; about 10⁵:1; about 10⁶:1; about 10⁷:1; about 10⁸:1; about10⁹:1; about 10¹⁰:1.

In some embodiments the ligand may be linked to a solid support such asa bead. A plurality of ligand linked beads may be used to reduce thenumber of extraneous phenotypic cells present in a mixed population ofcells. For example, a suitable number of beads may range from about 1bead per cell; about 5 beads per cell; about 10 beads per cell; about 15beads per cell; about 20 beads per cell; about 25 beads per cell; about30 beads per cell; about 35 beads per cell; about 40 beads per cell;about 45 beads per cell; about 50 beads per cell; about 60 beads percell; about 70 beads per cell; about 80 beads per cell; about 90 beadsper cell; about 100 beads per cell. To each bead in turn may be linkedto at least 10 ligands; at least 100 ligands; at least 1000 ligands; atleast 10,000 ligands; at least 10⁵ ligands; at least 10⁶ ligands; atleast 10⁷ ligands; at least 10⁸ ligands; at least 10⁹ ligands; at least10¹⁰ ligands; at least 10²⁰ ligands; at least 10³⁰ ligands at least 10⁴⁰ligands at least 10⁵⁰ ligands.

Typically the method of the invention comprises a step of co-incubatingthe ligand and the mixed cell population to facilitate binding of theligand to an extraneous phenotypic cell expressing a binding partner ofthe ligand. The ligand and mixed cell population may be incubated at atemperature ranging from about 3° C. to about 40° C. In some embodimentsthe ligand and the mixed cell population may be incubated at about 4°C.; about 20° C.; about 37° C. A suitable length of time for incubatingthe ligand and the mixed population of cells may range from about 1minute to about 60 minutes; about 5 minutes to about 50 minutes; about10 minutes to about 40 minutes. In some embodiments the ligand and mixedcell population may incubated for about 10 minutes; about 15 minutes;about 20 minutes; about 25 minutes; about 30 minutes; about 60 minutes.After contacting the mixed cell population with the ligand the cells maybe washed one or more times with a suitable buffer to wash awaynon-specifically bound ligand. Suitable buffers include PBS, anycommercially available isotonic buffer or media.

Separation of the ligand bound cells may be achieved by any method knownin the art for separating mixtures. For example the ligand may be taggedwith a detectable substance as described infra and separation may beachieved by cell sorting using a Fluorescent Activated Cell Sorter(FACS). As another example the ligand may be linked to a solid supportas described, infra. The ligand bound cells may be precipitated bygravity. Alternatively an external force may be applied to the ligandbound cells such that the ligand bound cells separate from the othercells comprising the mixed population of cells. For example, the ligandmay be linked to a magnetized solid support, such as a magnetic bead andthe mixed cell population including the ligand bound cells may beexposed to a magnetic field such that the ligand bound cells separatefrom the other cells comprising the mixed population of cells.

In other embodiments a cell may be contacted to with one or more ligandsthat bind specifically to a molecule expressed by a cell considered tobe of an extraneous phenotype. In some embodiments the ligand may beconjugated with a chemical agent before it is contacted with the cell.In other embodiments the ligand may be bound to the cell first and thencontacted with a chemical agent. Thus, the ligand bound cell may betargeted with a chemical agent that binds to the ligand bound cell,e.g., an agent that binds specifically to the ligand. In someembodiments one or more intervening ligands may be used. Thus a firstligand may bind the cell. A second ligand may bind the first ligand etc.The chemical agent may bind to any of the ligands bound to the cell. Theagent may be toxin and its binding to the ligand bound cell may therebykill the cell. Examples of suitable toxins include diphtheria toxin,tetanus toxin and the like. The agent may be a small molecule, aprotein, a peptide, a nucleic acid, a carbohydrate, a lipid. In oneembodiment where the ligand is an immunoglobulin the chemical agent maybe complement.

Where separation is performed, the extraneous phenotypic cell may beharvested by removal or elution from the ligand. Elution of theextraneous phenotypic cells may be achieved by altering one or more ofthe binding conditions such as the pH of the buffer containing the boundcells or the salt concentration of the buffer containing the boundcells.

Ligands

Any suitable ligand that binds specifically to a marker expressed by acell having an extraneous phenotype may be used. The ligand may becomprised of a protein or peptide fragment of a full length protein.Suitable proteins will include proteins or peptides that bindspecifically to a molecule expressed on the surface of the extraneousphenotypic cell; however in some embodiments the ligand may bind anintracellular target. The ligand may be also comprised of a nucleicacid, a sugar, a lipid, a glycoprotein, or a lectin or a combination ofany of these or a combination of a protein or peptide and any of these.Thus any ligand comprising a specific binding pair may be used as longas it binds specifically to a molecule expressed by the cells comprisingthe extraneous phenotype and does not bind a target phenotypic cell.

In some embodiments one or more ligands may be used. Thus a first ligandmay bind directly to a marker expressed by the extraneous phenotypiccell and a second ligand may then bind to the first ligand. Additionalligands which bind the second or any subsequent ligand are alsocontemplated. The second (or subsequent) ligand may comprise a tag whichfacilitates separation of the cells bound to the first ligand accordingto any method known in the art, e.g. cell sorting by FACS; precipitationof a ligand bound to a solid support; magnetic separation of ligandbound to a magnetic substrate; column chromatography over a ligandlinked to a solid support. Of course multiple molecules expressed by anextra-phenotypic cell may be bound each with a distinct ligand and eachof those ligands in turn may be contacted with one or more additionalligands.

In some embodiments the ligand may be an antibody. Antibody, as usedherein, includes an immunoglobulin or a part thereof, and encompassesany polypeptide comprising an antigen binding site regardless of thesource, method of production, and other characteristics. The termincludes for example, polyclonal, monoclonal, monospecific,polyspecific, humanized, single chain, chimeric, synthetic, recombinant,hybrid, mutated, and CDR grafted antibodies, as well as a moleculecomprised of two heavy and two light chains. A part of an antibody caninclude any fragment which can still bind antigen, for example, an Fab,F(ab′)2, Fv, scFv. In some embodiments the antibody may comprise avariable region which binds specifically to an epitope found on anextraneous phenotypic cell and at least a portion of an Fc region tofacilitate binding to a solid support or a second ligand. The antibodymay be linked to a solid support as described infra. In some embodimentsthe solid support may be bead. In specific embodiments the bead may bemagnetized.

In certain embodiments a suitable ligand may be an antibody to anepitope expressed on the surface of an epithelial cell or an epitheliallineage cell. The epitope may be comprised of proteins, carbohydrates,sugars, and/or lipids expressed on the surface of an epithelial cell.Suitable epitopes may be found on cells expressing one or more of thefollowing molecules: EpCAM; desmocollin, e.g. desmocollin 3; desmoglein,e.g. desmoglein 2; E-cadherin. Thus antibodies which bind to EpCAM,desmocollin, e.g. desmocollin 3, desmoglein, e.g. desmoglein 2,E-cadherin may be suitable ligands to practice the methods describedinfra. These antibodies may be used alone or in combination with one ormore additional ligands.

Additional ligands may include ligands that bind specifically tomolecules expressed by epithelial cells. Alternatively the ligands maybe combined with ligands that bind to one or more molecules expressed byundifferentiated pPS cells. For example antibodies that bind to epitopesfound on undifferentiated cells may be used in combination with ligandsthat bind to cells that express one or more markers found on epithelialcells. Thus examples of suitable antibodies that may be used in themethods for reducing the number of extraneous phenotypic cells in amixed population may include antibodies to TRA-1-60; TRA-1-81; SSEA 3;SSEA4; (See, Thomson 1998, Science 282:1145) Cripto, gastrin-releasingpeptide (GRP) receptor, and podocalyxin-like protein (see U.S. patentapplication Ser. No. 10/388,578) in addition to the ligands that bind toone or more markers expressed by epithelial cells.

The ligand may be coupled or linked covalently or non-covalently with adetectable substance to facilitate identification and/or isolation ofbound cells. A detectable substance may include any compound, which whenattached to a ligand, permits recognition of the presence of thisligand. The compound can comprise, for example, a radioactive molecule,a fluorescent molecule, a hapten, a carrier, an enzyme, an interveningmolecule such as biotin, or a dye. The detectable substance, forexample, may be a chemiluminescent material, or a bioluminescentmaterial. The ligand may also be linked with a toxin such as anymolecule that induces cell death, cell lysis etc.

In certain embodiments the ligand may be coupled or linked to a solidsupport. The solid support may comprise a bead, a gel, a monolith or amembrane. The solid support may comprise any material which can belinked to a ligand of interest (e.g., polystyrene, sepharose, sephadex).Solid supports may comprise any synthetic organic polymer such aspolyacrylic, vinyl polymers, acrylate, polymethacrylate, polyacrylamide,polyacylonitriles, and polyolefins. Solid supports may also comprise acarbohydrate polymer, e.g., agarose, cellulose, hyaluronic acid, chitin,acyl gellan, dextran, carboxymethylcellulose, carboxymethyl starch,carboxymethyl chitin, poly(lactide-co-ethylene glycol). Solid supportsmay comprise, for example, nitrocellulose, nylon, polyvinylidenefluoride (PVDF) or carboxylated polyvinylidene (U.S. Pat. No.6,037,124). The solid support may be coated with polyvinyl benzyldimethyl hydroxyethyl ammonium chloride, polyvinyl benzyl benzoylaminoethyl dimethyl ammonium chloride, polyvinyl benzyl tributylammonium chloride, copolymers of polyvinyl benzyl trihexyl ammoniumchloride and polyvinyl benzyl tributyl ammonium chloride, copolymers ofpolyvinyl benzyl dimethyl ammonium chloride and polyvinyl aminoethyldimethyl ammonium chloride (U.S. Pat. No. 5,336,596). Solid supports maycomprise inorganic oxides, such as silica, zirconia, e.g., carbon cladzirconia (U.S. Pat. No. 5,182,016), titania, ceria, alumina, manganese,magnesia (i.e., magnesium oxide), calcium oxide, controlled pore glass(CPG). Solid supports may also comprise combinations of some of theabove-mentioned supports including, but not limited to,dextran-acrylamide. A solid support may be prepared to minimizenon-specific interactions e.g., by coating it with one or more blockingproteins such as albumin, casein and the like. In some embodiments theligand may be linked to both a solid support and a detectable substance.

In some embodiments about the ligand may be linked to a solid supportsuch as a bead by mixing a suitable concentration of ligand and beadsunder conditions that facilitate covalent binding of the ligand to thesolid support. Suitable concentrations of ligand and beads range fromabout 0.1 mg of ligand/mL of beads to about 20 mg of ligand/mL of bead;from about 0.5 mg of ligand/mL of beads to about 10 mg of ligand/mL ofbead; from about 1.0 mg of ligand/mL of beads to about 5 mg of ligand/mLof bead. In some embodiments about 1 mg of ligand/mL of bead may beused. In other embodiments about 2 mg of ligand/mL of bead may be used.In some embodiments about 3 mg of ligand/mL of bead may be used. Inother embodiments about 4 mg of ligand/mL of bead may be used. In otherembodiments about 5 mg of ligand/mL of bead may be used.

In some embodiments an antibody may be linked directly to the bead usingknown conjugation chemistry such as the formation of EHS ester. In otherembodiments a specific binding partner of the antibody may be linkeddirectly to the bead and the antibody in turn may bind to the specificbinding partner thus linking it to the bead. The specific bindingpartner may bind to a region of the antibody that does not bind itsspecific epitope, e.g., a non variable region of the antibody. Asuitable region may include the Fc region of the antibody. Examples ofbinding partners that bind to the Fc region of an antibody includeprotein A and protein G. Alternatively, the antibody may be conjugatedto another molecule such as biotin and then coupled to a bead conjugatedwith streptavidin.

In one specific embodiment the ligand is an antibody to EpCAM linked,e.g., covalently to a bead such as a magnetic bead. The antibody may bedirectly linked to the bead or indirectly linked by an interveningmolecule such as protein A, protein G, biotin, streptavidin as describedin the previous paragraph. Alternatively, a short linker may serve toanchor the antibody to the bead thereby enhancing binding accessibilityof the antibody. The linker may be comprised of one or more amino acidsfor example.

In another specific embodiment the ligand may be an antibody to TRA-1-60linked covalently, e.g., to a bead such as a magnetic bead. The antibodymay be directly linked to the bead or indirectly linked by anintervening molecule such as protein A, protein G, biotin, streptavidinas described in the previous paragraph. Alternatively a short linker mayserve to anchor the antibody to the bead thereby enhancing bindingaccessibility of the antibody. The linker may be comprised of one ormore amino acids for example. The antibody to TRA-1-60 may have an IgGisotype in some embodiments. In other embodiments the antibody toTRA-1-60 may have an IgM isotype.

In some embodiments a combination of one or more antibodies to markersexpressed by epithelial cells may be used to deplete extraneousphenotypic cells from a mixed population of cells. In some embodimentsthe one more antibodies to markers expressed by epithelial cells may becombined with one or more antibodies to markers expressed byundifferentiated cells.

In some embodiments a combination of one or more antibodies to EpCAM andone or more antibodies to TRA-1-60 may be used to deplete extraneousphenotypic cells from a mixed population of cells. In some embodimentsall of the antibodies used may have an IgG isotype. In other embodimentsat least one of the antibodies may have an IgG isotype. In still otherembodiments at least one of the antibodies may have an IgM isotype.

Cell Populations

In certain embodiments the invention provides a mixed population ofcells that is enriched for a targeted phenotype. The mixed population ofcells may be enriched for a targeted cell type by eliminating at leastone cell having an extraneous phenotype from the mixed population ofcells.

1. Extraneous Phenotypes

In some embodiments extraneous phenotypic cells may include for examplea cell having an epithelial morphology, a cell expressing at least onemarker expressed in or on an epithelial cell, a cell capable of formingan clustered epithelial structure when implanted into a subject, anundifferentiated pluripotent cell, a cell having the morphology of anundifferentiated cell, a cell expressing at least one marker expressedin or on undifferentiated cells. Examples of markers expressed in or onundifferentiated TRA-1-60, TRA-1-81; SSEA 3; SSEA 4; Oct 4. Examples ofmarkers expressed in or on epithelial cells include EpCAM, andcytokeratin. Other suitable markers expressed on epithelial cells mayinclude desmocollin, desmoglein, and E-cadherin. These cells may havethe capacity to form epithelial clusters when implanted into a subjectsuch as a rodent.

2. Targeted Phenotypes

Targeted phenotypic cells may be the in vitro differentiated progeny ofpPS cells and may include any of the following: oligodendrocytes, neuralcells such as neurons and astrocytes, cardiomyocytes, hematopoieticcells, pancreatic islet cells, hepatocytes, osteoblast and chondrocytes.In some embodiments the target cell may be a mature target cell such asa mature cardiomyocyte, a mature oligodendrocyte, etc. In otherembodiments the targeted cell type may a precursor cell such as acardiomyocyte precursor, an oligodendrocyte precursor, a neural cellprecursor, an islet cell precursor, a hematopoietic cell precursor, ahepatocyte precursor, an osteoblast precursor, a chondrocyte precursor.Precursor cells may express one or more markers expressed by acorresponding mature cell type; and/or have one or more morphologicalfeatures found on the corresponding mature cell types and/or have theability to differentiate into the corresponding mature cell type eitherin vivo or in vitro. Targeted cell types may include cells that expressone or more markers expressed on, in or by one of the following celltypes: oligodendrocytes, neuronal cells cardiomyocytes, hematopoieticcells, pancreatic islet cells, hepatocytes, osteoblasts and chondrocytes(examples of suitable markers are provided in the paragraphs thatfollow). Markers may be detected using any method known in the art, forexample immunocytochemistry, immunohistochemistry, FACS, western blot,ELISA, or qPCR may be used to detect one or more markers expressed on,in or by a target cell.

Methods of differentiating pPS cells in vitro into oligodendrocytes havebeen described in U.S. Pat. No. 7,285,415, which is hereby incorporatedby reference in its entirety. Oligodendrocytes may be differentiated invitro from a population of pPS cells for example by contacting the pPScells with a growth factor, a ligand to a thyroid hormone receptor and aligand for a retinoic acid receptor. Markers expressed on, in or byoligodendrocytes include NG2, a chondroitin sulfate proteoglycanexpressed by macrophages and oligodendrocyte progenitors;galactocerebroside (GalC), a marker for committed oligodendrocytes;myelin basic protein (MBP), a marker of mature myelin; microtubuleassociated protein 2 (MAP-2), a marker for CNS cells; myelin proteolipidprotein, a component of myelin that is expressed on oligodendrocytes andglial precursors; the epitope defined by the 04 antibody, a marker foroligodendrocytes, astrocytes, and their precursors; A2B5, an epitopeexpressed on type 2 astrocytes, glial progenitors, and oligodendrocyteprogenitors; the epitope recognized by RIP antibody, which stainsoligodendrocytes and their processes, and coincides with myelinatedaxons in both the spinal cord and the cerebellum.

Methods of differentiating pPS cells in vitro into neural cells havebeen described, see, e.g., U.S. Pat. No. 6,833,269; U.S. PatentPublication Nos. 2005/0095707; 2005/0158855, all of which areincorporated by reference in their entirety. Neural cells may bedifferentiated in vitro from a population of PS cells for example bycontacting the pPS cells with noggin and/or follistatin; by contactingthe pPS cells with a TGF β antagonist or by forming embryoid bodies(EBs) and culturing the EBs in suspension with 10 μM retinoic acid, thenplating the cells into defined medium supplemented with epidermal growthfactor (EGF), fibroblast growth factor (bFGF), platelet derived growthfactor (PDGF), and insulin like growth factor 1 (IGF-1). Markersexpressed on, in or by neural cells include NCAM, A2B5, β tubulin III,and microtubule-associated protein 2 (MAP 2).

Methods of differentiating pPS in vitro into cardiomyocytes have beendescribed, see, e.g., U.S. Pat. Nos. 7,452,718; 7,425,448; U.S. PatentPublication Nos. 2005/0054092; 2007/0010012, all of which are herebyincorporated by reference in their entirety. pPS cells may be contactedwith activin followed by bone morphogenic protein (BMP) to differentiatepPS cells into cardiomyocytes. Markers expressed on, in or bycardiomyocytes include cardiac troponin I (cTnI), a subunit of troponincomplex that provides a calcium sensitive molecular switch for theregulation of striated muscle contraction; cardiac troponin T (cTnT);Nkx2.5, a cardiac transcription factor expressed in cardiac mesodermduring early embryonic development, which persists in the developingheart; atrial natriuretic factor (ANF), a hormone expressed indeveloping heart and fetal cardiomyocytes but down-regulated in adults.Other suitable markers include myosin heavy chain (MHC), particularlythe β chain which is cardiac specific; titin, tropomyosin, a sarcomericactinin, and desmin; GATA-4, a transcription factor that is highlyexpressed in cardiac mesoderm and persists in the developing heart. Yetother suitable markers may include MEF 2A, MEF 2B, MEF 2C, MEF 2D;N-cadherin, which mediates adhesion among cardiac cells; connexin 43,which forms the gap junction between cardiomyocytes; β1 adrenoceptor (β1AR); creatine kinase MB (CK MB) and myoglobin, which are elevated inserum following myocardial infarction; a cardiac actin.

Methods of differentiating pPS cells in vitro into hematopoietic cellshave been described see, e.g., U.S. Pat. No. 7,247,480; U.S. patentapplication Ser. No. 12/412,480 and U.S. Patent Publication No.2005/0282272, all of which are hereby incorporated by reference in theirentirety. As an example pPS cells may be differentiated into dendriticcells by contacting the pPS cells with a plurality of cytokines such asBMP-4, granulocyte macrophage colony stimulating factor (GM-CSF), stemcell factor (SCF) and vascular endothelial growth factor (VEGF) todifferentiate the pPS cells into immature dendritic cells. The immaturedendritic cells may then be contacted with a maturation cocktail e.g.,GM-CSF, tumor necrosis factor α (TNFα), interleukin 1-β (IL-1β),interferon γ (IFNγ), and prostaglandin E2 (PGE2) to generate maturedendritic cells. Markers expressed in on or by hematopoietic cellsinclude CD34, CD59, Thy1/CD90, CD38, C-kit/CD117, CD13, IL-3Rα (CD123),and CD45RA, CD64 CD14, CD45, CD11a, CD11b, CD15 CD11c, MHC I, MHC II,CD83, CCR7, CD205, CD86, CD40, MHC I, MHC II, CD8, CD4, CD119, CD74,CD71, CD75, CD51, CD56, CD65, CD10, CD66a, CD66b, CD44, betaglobin andmyoglobin.

Methods of differentiating pPS cells in vitro into islet cells have beendescribed, see, e.g., U.S. Pat. No. 7,033,831 and U.S. patentapplication Ser. No. 12/303,895, both of which are incorporated byreference in their entirety. As an example pPS cells may bedifferentiated into islet cells by first contacting the pPS cells withactivin and sodium butyrate followed by noggin, fibroblast growth factor(FGF), epidermal growth factor (EGF), insulin like growth factor 2(IGF-2) and nicotinamide. Markers expressed in on or by islet cells(including islet cell precursors) include insulin, c-peptide, Hlxb9,Pdx1, Neurogenin3, Pax4, NeuroD1, Isl1, Nkx2.2, and Nkx6.1; pancreaticpolypeptide, islet amyloid polypeptide (IAPP), Islet 1, Beta 2/NeuroD,HNF4a. Also included are definitive endoderm cells, a particular isletprecursor cell that express Sox17 and HNF3β.

Methods of differentiating pPS cells in vitro into hepatocytes have beendescribed, see, e.g., U.S. Pat. Nos. 6,458,589; 6,506,574; 7,256,042;7,473,555; and U.S. patent application Ser. No. 12/303,104, all of whichare incorporated by reference in their entirety. For example pPS cellsmay be differentiated in vitro into hepatocytes by contacting the pPScells with DMSO and butyrate followed by EGF, TGF-α, hepatocyte growthfactor (HGF), and butyrate. Markers expressed in on or by hepatocytesinclude α₁-antitrypsin (AAT), albumin; asialoglycoprotein receptor(either the ASGR-1 or ASGR-2 isotype); Ck8, Ck18, Ck19, cytochrome p450,CYP1A1/2d CYP2A6 CYP2B6, CYP2C9, CYP2E1, CYP3A3-5 glucose-6-phosphatase;HNF-4α, α-fetoprotein, apoE, glucokinase, insulin like growth factors 1and 2 receptor, insulin receptor, leptin, apoAI, apoAII, apoB, apoCIII,apoCII, aldolase B, phenylalanine hydroxylase, L-type fatty acid bindingprotein, transferrin, retinol binding protein, erythropoietin (EPO), andNADPH-Cc.

Methods of differentiating pPS cells in vitro into osteoblasts have beendescribed, see, e.g., U.S. Patent Application No. 2005/028274, which isincorporated by reference in its entirety. For example pPS cells may bedifferentiated in vitro into osteoblasts by contacting the pPS cellswith bone morphogenic protein (BMP), a ligand for the human TGF-3receptor, or a ligand for the vitamin D receptor. The cells may also becontacted with dexamethasone, ascorbic acid-2-phosphate, and sources ofcalcium and phosphate. Markers expressed in on or by osteoblasts includeosteocalcin, osteonectin, type 1 collagen, alkaline phosphatase, BMPreceptors, PTH receptors, CD105 (endoglin), CD106 (VCAM), CD166 (ALCAM),CD29, CD44 and GATA 4.

Methods of differentiating pPS cells in vitro into chondrocytes havebeen described, see, e.g., U.S. Patent Application No 2006/0148077. Forexample pPS cells may be differentiated in vitro into chondrocytes bycontacting the pPS cells with transforming growth factors (TGF),fibroblast growth factors (FGF), growth and differentiation factors(GDF), bone morphogenic proteins (BMP), hedgehog proteins (HH),L-ascorbic acid, and parathyroid hormone-related protein (PTHrP). Thecells may be maintained in culture during the differentiation process asa micromass. Markers express in on or by chondrocytes include Type IIcollagen or aggrecan.

Enriched Cell Populations

As discussed above the invention provides cell populations enriched fora target phenotype. The cell populations may be enriched by reducing thenumber of extraneous phenotypic cells from the mixed population ofcells. The extraneous phenotypic cell may express one or more markersexpressed by epithelial cells as discussed infra. The extraneousphenotypic cells may have the morphology of epithelial cells. Theextraneous phenotypic cells may have the capability of forming aclustered epithelial structure when implanted into a subject. Theextraneous phenotypic cells may be undifferentiated pPS cells. Theextraneous phenotypic cells may be cells that express one or moremarkers expressed by a pPS cell, e.g., TRA-1-60, TRA-1-81, Oct4, SSEA3,SSEA4. The extraneous phenotypic cells may comprise a combination of oneor more of the extraneous phenotypic cells described in this paragraph.Thus in certain embodiments the invention provides enriched populationsof cell comprising a targeted phenotype wherein at least 1%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99% of theextraneous phenotypic cells have been removed from the cell populationcomprising the target cell phenotype.

In other embodiments the invention provides a mixed cell populationcomprising a targeted phenotype, wherein at least one cell having anextraneous phenotype has been removed from the mixed population, andwherein the target phenotype comprises at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, of the cells in thepopulation after the at least one cell having an extraneous phenotypehas been removed. The targeted cell type may be chosen fromoligodendrocytes, neural cells, cardiomyocytes, hematopoietic cells,pancreatic islet cells, hepatocytes, osteoblast and chondrocytes or froma cell that expresses one or more markers expressed on, in or by a cellchosen from oligodendrocytes, neuronal cells, cardiomyocytes,hematopoietic cells, pancreatic islet cells, hepatocytes, osteoblast andchondrocytes.

Kits

In certain embodiments the invention provides a kit for depleting cellshaving an extraneous phenotype from a mixed population of cells whereinthe mixed population of cells comprises a targeted phenotypic cell andextraneous phenotypic cells. Both cell types may be the in vitro progenyof pPS cells. For example the targeted phenotypic cells may be comprisedof the in vitro differentiated progeny of the pPS cells while theextraneous phenotypic cells may comprise differentiated progeny of thepPS cells, undifferentiated progeny of the pPS cells or a combination ofboth. The kit may comprise one or more ligands that specifically bind toone or more molecules expressed by the cells comprising the extraneousphenotype. The one or more ligands may be provided in one or morecontainers. The ligands may be provided in a solution such as an aqueousbuffer, e.g., an isotonic buffer, PBS or the like. Alternatively the oneor more ligands may be provided in lyophilized form. The kit may includeinstructions for reconstituting the lyophilized ligand. The kit mayinclude instructions for contacting the mixed population of cells withthe ligand. The instructions may include a suitable concentration ofligand to use to deplete the extraneous phenotype. Optionally the kitmay comprise one or more controls, e.g., a positive control cell typethat will bind to the one or more ligands provided and a negativecontrol cell type that will not bind to the one or more providedligands.

In some embodiments the ligand provided in the kit may be an antibody.The ligand may be an antibody that binds to a molecule expressed by acell comprising the extraneous phenotype. Examples of suitableantibodies include a TRA-1-60 antibody, an EpCAM antibody, a cytokeratinantibody such as a pan-cytokeratin antibody, an antibody to TRA-1-81, anantibody to SSEA3, and an antibody to SSEA4. The antibody may be anyisotype e.g., IgG, IgM, IgA, IgE, IgD. In one embodiment the antibodymay be an IgG that binds to EpCAM. In one embodiment the antibody may bean IgG that binds to a cytokeratin. In one embodiment a plurality of IgGantibodies binding to different epitopes on a plurality of cytokeratinsmay be used. In one embodiment the antibody may be an IgM that binds toTRA-1-60. In one embodiment the antibody may be an IgG that binds toTRA-1-60. In one embodiment a combination of different antibodies may beused, e.g., a plurality of the types of antibodies described in thisparagraph. Thus a plurality of antibodies directed to a single antigen(but different epitopes) may be used. A plurality of antibodies directedto a plurality of antigens may be used.

The kit may comprise a solid support for the ligand. Solid supports aredescribed in detail infra. The ligand may be provided already linked tothe solid support in a single container. Alternatively the kit mayprovide the antibody and solid support unlinked to one another. Thesolid support and the ligand may thus be provided in separatecontainers. The kit may provide instructions and one or more reagentsfor linking the ligand to the solid support.

The kit may further comprise one or more detectable substances which maybe linked to the one or more ligands provided in the kit. Detectablesubstances are described in detail infra. The one or more detectablesubstances may each be provided in one or more separate containers alongwith instructions for linking the one or more detectable substances tothe one or more ligands.

The kit may provide a means for applying an external force to the mixedpopulation of cells after it has been contacted with the ligand bound toa solid support such that separation of the ligand bound extraneousphenotypic cells from the mixed population of cells is facilitated. Theexternal force may be a magnetic field.

In one embodiment the kit may comprise one or more antibodies chosenfrom an antibody to EpCAM, an antibody to cytokeratin, e.g.,pan-cytokeratin, an antibody to desmocollin, an antibody to desmoglein,and an antibody to E-cadherin and/or an antibody to TRA-1-60. The one ormore antibodies may be conjugated to a magnetic bead. The kit mayfurther comprise a magnetic field source adapted to facilitate removalof the extraneous phenotypic cells bound to the antibodies. The kit maycomprise one or more containers to hold the one or more antibodies andthe source of the magnetic field. The kit may optionally include washsolutions and/or buffers and instructions for using the antibodies andthe magnetic field.

Uses of Enriched Cell Populations

This invention provides a method to produce large numbers of cellsenriched for a target phenotype. Target phenotypes includeoligodendrocytes, neural cells, cardiomyocyte, hematopoietic cells,islet cells, hepatocytes, chondrocytes and osteoblasts. These cellpopulations can be used for a number of important research, development,and commercial purposes. Because the populations provided are enrichedfor a target phenotype they will be more suitable for all of the usesdescribed infra when compared to cell populations that have not been soenriched.

Screening

The enriched target cell populations of this invention can be usedcommercially to screen for factors (such as solvents, small moleculedrugs, peptides, oligonucleotides) or environmental conditions (such asculture conditions or manipulation) that affect the characteristics ofsuch cells and their various progeny. Characteristics may includephenotypic or functional traits of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on enriched target cellpopulations. Screening may be done either because the compound isdesigned to have a pharmacological effect on the cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on cells of the target phenotype. Other screening applicationscould include screening compounds for carcinogenic or other toxiceffects. The screening can be conducted using any of the precursor cellsor terminally differentiated/mature cells of the invention in order todetermine if the target compound has a beneficial or harmful effect onthe target cell. Using the enriched target populations described infrawill provide for more accurate screening results.

The reader is referred generally to the standard textbook In vitroMethods in Pharmaceutical Research, Academic Press, 1997. Assessment ofthe activity of candidate pharmaceutical compounds generally involvescombining the enriched target cells of this invention with the candidatecompound, either alone or in combination with other drugs. Theinvestigator determines any change in the morphology, marker phenotypeas described infra, or functional activity of the cells, that isattributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlates the effect of thecompound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in In vitro Methods in PharmaceuticalResearch, Academic Press, 1997) for further elaboration.

Where an effect is observed, the concentration of the compound can betitrated to determine the median effective dose (ED₅₀).

Animal Testing

This invention also provides for the use of enriched target cells of theinvention to enhance tissue maintenance or repair of tissue function forany perceived need, such as an inborn error in metabolic function, theeffect of a disease condition, or the result of significant trauma.

To determine the suitability of cell compositions for therapeuticadministration, the enriched target cells can first be tested in asuitable animal model such as a rat, mouse, guinea pig, rabbit, cow,horse, sheep, pig, dog, primate or other mammal. At one level, cells areassessed for their ability to survive and maintain their phenotype invivo. Cell compositions may be administered to immunodeficient animals(such as nude mice, or animals rendered immunodeficient chemically or byirradiation). Tissues are harvested after a period of regrowth, andassessed as to whether pluripotent stem derived cells are still present.Functional tests as are known in the art may be performed.

Cell survival may be monitored by administering cells that express adetectable label (such as green fluorescent protein, orβ-galactosidase); that have been prelabeled (for example, with BrdU or[³H]thymidine), or by subsequent detection of a constitutive cell marker(for example, using human-specific antibody). The presence and phenotypeof the administered cells can be assessed by immunohistochemistry orELISA using human-specific antibody, or by RT-PCR analysis using primersand hybridization conditions that cause amplification to be specific forhuman polynucleotides, according to published sequence data.

In one embodiment the enriched target cell populations may beadministered to an animal to test for the formation of clusteredepithelial structures. The animal may be a rodent such as a mouse or arat. The enriched cells may be administered to the animal for a suitablelength of time, e.g., about 1-12 months. The animal may be sacrificedand the tissue receiving the enriched cell population may be subjectedto histological examination or ICC or both to screen for primate cellshaving epithelial morphology and/or labeling for one or more markersfound on an epithelial cell and/or one or more markers found on anundifferentiated pPS cell.

In other embodiments the enriched target cells of the invention may betested functionally using a known animal model for a particular disease.If the target cell type is a cardiomyocyte, for example, one of severalmodels for heart disease and or infarction can be used. Hearts can becryoinjured by placing a precooled aluminum rod in contact with thesurface of the anterior left ventricle wall (Murry et al., J. Clin.Invest. 98:2209, 1996; Reinecke et al., Circulation 100:193, 1999; U.S.Pat. No. 6,099,832; Reinecke et al., Circ Res., Epub March 2004). Inlarger animals, cryoinjury can be effected by placing a 30-50 mm copperdisk probe cooled in liquid N₂ on the anterior wall of the leftventricle for ˜20 min (Chiu et al., Ann. Thorac. Surg. 60:12, 1995).Infarction can be induced by ligating the left main coronary artery (Liet al., J. Clin. Invest. 100:1991, 1997) or by using an ameroidconstriction device that gradually swells to occlude an artery. Injuredsites are treated with cell preparations of this invention, and theheart tissue is examined by histology for the presence of the cells inthe damaged area. Cardiac function can be monitored by determining suchparameters as left ventricular end-diastolic pressure, developedpressure, rate of pressure rise, and rate of pressure decay.

Where the target cell is an oligodendrocyte, regions of chronicdemyelination may be induced in the adult rat dorsal column (Keirsteadet al., J Neurosci. 19:7529, 1999). The spinal cord may be exposed to 40G of X irradiation over a distance of 2 cm centered on T9 using leadshielding, which introduces nicks into the DNA of exposed cells, thuscausing death of the cells that are dividing. This is followed by directintraspinal injection of ethidium bromide 2 days later at T9. Ethidiumbromide is a DNA interchelating agent that kills cells exposed to it,rendering an acellular region of chronic demyelination that is free ofviable oligodendrocytes and astrocytes through ˜60% of the area of thedorsal column.

Three days later, the animals receive transplants of enrichedpopulations of oligodendrocyte lineage cells into the site ofdemyelination. Optionally, the cells can be prelabeled withbromodeoxyuridine (BrdU) added to the culture medium 48 hours inadvance. In the first instance, cells can be prepared as clusters of ˜30precursors, concentrated to a density of ˜60,000 cells per μL. One μL ofcells is administered into the injury site using a pulled glassmicropipette or syringe needle of about 80 um outside diameter over aperiod of about 10 minutes. After about 2-4 weeks, tissue samples areprepared for resin or cryostat sectioning into 1 mm transverse blocks.

Sections from the coronal face are stained with toluidine blue andanalyzed for general pathology, evidence of remyelination, and cellmorphology. Since the damaged regions are acellular, cells present aftertransplantation are derived from the administered cells. Cryostatsections can be stained for markers of relevant cell types, such as GFAP(astrocytes), CNP (oligodendrocytes), RIP (oligodendrocytes), or NeuN(neurons). Ultrathin sections can also be analyzed by electronmicroscopy for the number of myelin lamellae and cellularultrastructure. Redistribution of transplanted cells throughout areasdemyelination, and differentiation into mature myelinating cells can bedetermined. Remyelination as a percentage of demyelinated axons at alevel of 25%, 50%, or 75% is evidence of increased biological efficacy.

The enriched target cells can be tested in a model for SCI includingcontusion injuries and dorsal hemisection. For contusion injuries, thespinal course is displaced for about 0.9 mm (moderate injury) over 23milliseconds using a suitable spinal contusion injury device. Forhemisection injuries, the dorsal half of the spinal cord is cut with apointed scalpel blade using a stereotactic manipulator. Both proceduresare followed by suitable postoperative care. To promote migration ofimplanted cells and remove myelin-associated growth inhibitors, thespinal cord may optionally also be demyelinated (Keirstead et al., ExpNeurol. 151:303, 1998). A 2 mm hole is produced in the center of thevertebra canal and caudal to the site of axonal injury. The exposedspinal cord is the injected with about 4 μL polyclonal anti GalCantibody (Millipore Corp., Billerica, Mass.) at a dilution of 1:2 with33% guinea pig complement (Harlan SeraLab, San Francisco, Calif.) inphosphate buffered saline.

The animals are transplanted with enriched target phenotypeoligodendrocyte lineage cells about 24 hours after injury through apulled glass micropipette or syringe needle. Alternatively, a chronicinjury model can be created by withholding treatment after injury for1-3 months. Following treatment with the cells, functional response canbe recorded by video tape, and monitored on a regular basis for evidenceof clinical improvement. For example, overground locomotion can bequantitated using the BBB scale, a 21-point scale based on jointmovements, weight support, limb coordination, and other features.

Therapeutic Use in Humans

After adequate testing, enriched target cells of this invention can beused for tissue reconstitution or regeneration in a human patient orother subject in need of such treatment. The cells are administered in amanner that permits them to graft or migrate to the intended tissue siteand reconstitute or regenerate the functionally deficient area. Thusenriched target cell populations comprising cardiomyocytes may beadministered to the heart. Enriched target cell populations comprisingoligodendrocytes may be administered to the spinal cord. Enriched targetcell populations comprising hepatocytes may be administered to theliver. Enriched target cell populations comprising hematopoietic cellsmay be administered intravenously. Enriched populations of cellscomprising islet cells may be administered near the kidney or pancreas.Alternatively, the enriched population of cells comprising islet cellsmay be administered subcutaneously in an implantable device that shieldsthe cells from an auto-immune response. Enriched populations of cellscomprising chondrocytes may be administered to a joint that is deficientin cartilage or in need of cartilage repair. Enriched populations ofosteoblasts may be administered to a fractured bone.

Administration of the population of cells may be achieved by any methodknown in the art. For example the cells may be administered surgicallydirectly to the organ or tissue in need of a cellular transplant.Alternatively non-invasive procedures may be used to administer thecells to the subject. Examples of non-invasive delivery methods includethe use of syringes and/or catheters to deliver the cells into the organor tissue in need of cellular therapy.

The patient receiving an allograft of enriched target cells of theinvention may be treated to reduce immune rejection of the transplantedcells. Methods contemplated include the administration of traditionalimmunosuppressive drugs like tacrolimus, cyclosporin A (Dunn et al.,Drugs 61:1957, 2001), or inducing immunotolerance using a matchedpopulation of pluripotent stem derived cells (WO 02/44343; U.S. Pat. No.6,280,718; WO 03/050251). Alternatively a combination ofanti-inflammatory (such as prednisone) and immunosuppressive drugs maybe used.

The enriched target cells of this invention can be supplied in the formof a pharmaceutical composition, comprising an isotonic excipientprepared under sufficiently sterile conditions for human administration.To reduce the risk of cell death upon engraftment, the cells may betreated by heat shock or cultured with ˜0.5 U/mL erythropoietin ˜24hours before administration.

For general principles in medicinal formulation, the reader is referredto Cell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan, Eds., Cambridge UniversityPress, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000. Choice of the cellular excipientand any accompanying elements of the composition will be adapted inaccordance with the route and device used for administration. Thecomposition may also comprise or be accompanied with one or more otheringredients that facilitate the engraftment or functional mobilizationof the enriched target cells. Suitable ingredients will vary dependingon the target phenotype but may include matrix proteins that support orpromote adhesion of the target cell type or that promote vascularizationof the implanted tissue.

This invention also includes a reagent system, comprising a set orcombination of cells that exist at any time during manufacture,distribution, or use. The cell sets comprise any combination of two ormore cell populations described in this disclosure, exemplified but notlimited to a type of differentiated pluripotent stem-derived cell, incombination with undifferentiated primate pluripotent stem cells orother differentiated cell types, often sharing the same genome. Eachcell type in the set may be packaged together, or in separate containersin the same facility, or at different locations, at the same ordifferent times, under control of the same entity or different entitiessharing a business relationship.

Pharmaceutical compositions of this invention may optionally be packagedin a suitable container with written instructions for a desired purpose,such as the reconstitution of cardiomyocyte-lineage cell function toimprove a disease condition or abnormality of the cardiac muscle orremylination of an injured spinal cord.

The enriched target cells of this invention can be used to prepare acDNA library relatively uncontaminated with cDNA preferentiallyexpressed in cells from other lineages. For example, enriched targetcells are collected by centrifugation at 1000 rpm for 5 minutes, andthen mRNA is prepared and reverse transcribed. Expression patterns ofthe enriched target cells may be compared with other cell types bymicroarray analysis, reviewed generally by Fritz et al. Science 288:316,2000. Such libraries would be especially well suited for studying geneexpression in target cells compared to the undifferentiated pPS cellsfrom which they were derived. Because these cells share essentiallyidentical genomes comparisons in gene expression using for examplesubtractive hybridization can be made with little or no backgroundnoise. Reducing the number of extraneous phenotypic cells within a cellpopulation will provide improved signal to noise ratios in comparinggene expression in two populations of cells, e.g., differentiated targetprogeny and the parent pPS cell line giving rise to the differentiatedtarget cells.

The differentiated cells of this invention can also be used to prepareantibodies that are specific for markers of the enriched target cells.Polyclonal antibodies can be prepared by injecting a vertebrate animalwith cells of this invention in an immunogenic form. Production ofmonoclonal antibodies is described in such standard references as Harlow& Lane (1988) Antibodies: A Laboratory Manual, U.S. Pat. Nos. 4,491,632,4,472,500 and 4,444,887, and Methods in Enzymology 73B:3 (1981).

Primate Pluripotent Stem Cells

The present invention provides methods for enriching target cellsdifferentiated in vitro from pPS cells. pPS cells include any primatepluripotent cell. A pluripotent cell will, under appropriate growthconditions, be able to form at least one cell type from each of thethree primary germ layers: mesoderm, endoderm and ectoderm. The pPScells may originate from pre-embryonic, embryonic or fetal tissue ormature differentiated cells. Typically, the pPS cells are not derivedfrom a malignant source. pPS cells will form teratomas when implanted inan immuno-deficient mouse, e.g., a SCID mouse. The pPS cells may beobtained from an established cell line. Established cell lines areavailable from public cell banks such as WiCell and the UK Stem CellBank.

Under the microscope, pPS cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with poorlydiscernable cell junctions. pPS cells typically express thestage-specific embryonic antigens (SSEA) 3 and 4, and markers detectableusing antibodies designated TRA-1-60 and TRA-1-81. Undifferentiatedhuman embryonic stem cells also typically express the transcriptionfactor Oct-3/4, Cripto, gastrin-releasing peptide (GRP) receptor,podocalyxin-like protein (PODXL), nanog and telomerase reversetranscriptase, e.g., hTERT (US 2003/0224411 A1), as detected by RT-PCR.

pPS cells that may be used in any of the embodiments of the inventioninclude, but are not limited to, embryonic stem cells such as humanembryonic stem cells (hES). Embryonic stem cells used in the inventionmay be chosen from embryonic stem cell lines. A large number ofembryonic stem cell lines have been established including, but notlimited to, H1, H7, H9, H13 or H14 (Thompson, (1998) Science 282:1145);hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen, Inc., Athens, Ga.); HES-1,HES-2, HES-3, HES-4, HES-5, HES-6 (from ES Cell International, Inc.,Singapore); HSF-1, HSF-6 (from University of California at SanFrancisco); I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3, J 3.2 (derived atthe Technion-Israel Institute of Technology, Haifa, Israel); UCSF-1 andUCSF-2 (Genbacev et al., (2005) Fertil. Steril. 83(5):1517); lines HUES1-17 (Cowan et al., (2004) NEJM 350(13):1353); and line ACT-14(Klimanskaya Ct al., (2005) Lancet, 365(9471):1636).

Other primate pluripotent stem cell types include, but are not limitedto, primitive ectoderm-like (EPL) cells, described in WO 01/51610 andhuman embryonic germ (hEG) cells (Shamblott et al., (1998) Proc. Natl.Acad. Sci. USA 95:13726).

pPS cells suitable for use in any of the embodiments of the inventionalso include induced primate pluripotent stem (iPS) cells. iPS cellsrefer to cells that are genetically modified, e.g., by transfection withone or more appropriate vectors, such that they attain the phenotype ofa pPS cell (Takahashi et al. (2007) Cell 131(5):861; Yu et al. (2007)Science 318:1917). Alternatively, iPS cells may be obtained byreprogramming adult cells by contacting them with a protein cocktailthat induces the cells to reprogram such that they have phenotypic andmorphological traits associated with blastocyst derived pluripotent stemcells see, Kim et al. (2009) Cell Stem Cell 4(6):472. Phenotypic traitsattained by these reprogrammed cells include morphology resemblingpluripotent stem cells isolated from a blastocyst, as well as expressionof surface antigens, gene expression and telomerase activity found inpPS cells. The iPS cells may have the ability to differentiate into atleast one cell type from each of the primary germ layers: ectoderm,endoderm and mesoderm. The iPS cells may also form teratomas wheninjected into immunodeficient mice, e.g., SCID mice. (Takahashi et al.,(2007) Cell 131(5):861; Yu et al., (2007) Science 318:1917).

Culture Conditions for Primate Pluripotent Stem Cells

In certain embodiments, pPS cells used in the present invention may havebeen derived in a feeder-free manner (see, e.g., Klimanskaya et al.,(2005) Lancet 365(9471):1636). In certain embodiments the pPS may becultured prior to use in a serum free environment.

pPS cells may be cultured using a variety of substrates, media, andother supplements and factors known in the art. In some embodiments asuitable substrate may be comprised of a matrix including one or more ofthe following: laminin, collagen, fibronectin, vitronectin, heparinsulfate proteoglycan. In some embodiments the matrix may comprise asoluble extract of the basement membrane from a murine EHS sarcoma whichis commercially available as Matrigel™ (BD Biosciences, San Jose,Calif.). In other embodiments the matrix may comprise one more isolatedmatrix proteins of human, humanized, or murine origin, e.g., CELLstart™(Invitrogen, Carlsbad, Calif.). In still other embodiments a suitablesubstrate may be comprised of one or more polymers such as one or moreacrylates. The polymers may include one or more proteins or peptidefragments derived from a protein found in vivo in the extra-cellularmatrix. In one particular embodiment the substrate is comprised of oneor more acrylates and a conjugated vitronectin peptide (see, e.g. U.SPatent Publication No. 2009/0191633; U.S Patent Publication No.2009/0191626; U.S Patent Publication No. 2009/0203065). pPS cells can bepropagated continuously in culture, using culture conditions thatpromote proliferation while inhibiting differentiation.

Exemplary medium may be made with 80% DMEM (such as Knock-Out DMEM,Gibco), 20% of either defined fetal bovine serum (FBS, Hyclone) or serumreplacement (US 2002/0076747 A1, Life Technologies Inc.), 1%non-essential amino acids, 1 mM L-glutamine, and 0.1 mM0-mercaptoethanol. Other suitable media include serum free defined mediasuch as X-VIVO™ 10 (Lonza, Walkersville, Md.). Still other commerciallyavailable media formulations that may be used in certain embodiments ofthe invention include X-VIVO™ 15 (Lonza, Walkersville, Md.); mTeSR™(Stem Cell Technologies, Vancouver, Calif.); hTeSR™ (Stem CellTechnologies, Vancouver, Calif.), StemPro™ (Invitrogen, Carlsbad,Calif.) and Cellgro™ DC (Mediatech, Inc., Manassas, Va.).

In certain embodiments, pPS cells may be maintained in anundifferentiated state without added feeder cells (see, e.g., (2004)Rosier et al., Dev. Dynam. 229:259). Feeder-free cultures are typicallysupported by a nutrient medium containing factors that promoteproliferation of the cells without differentiation (see, e.g., U.S. Pat.No. 6,800,480). In certain embodiments, conditioned media containingsuch factors may be used. Conditioned media may be obtained by culturingthe media with cells secreting such factors. Suitable cells includeirradiated (˜4,000 rad) primary mouse embryonic fibroblasts, telomerizedmouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S.Pat. No. 6,642,048). Medium can be conditioned by plating the feeders ina serum free medium such as KO DMEM supplemented with 20% serumreplacement and 4 ng/mL bFGF. Medium that has been conditioned for 1-2days may be supplemented with further bFGF, and used to support pPS cellculture for 1-2 days (see. e.g., WO 01/51616; Xu et al., (2001) Nat.Biotechnol. 19:971).

Alternatively, fresh or non-conditioned medium can be used, which hasbeen supplemented with added factors (like a fibroblast growth factor orforskolin) that promote proliferation of the cells in anundifferentiated form. Exemplary is a base medium like X-VIVO™ 10(Lonza, Walkersville, Md.) or QBSF™-60 (Quality Biological Inc.Gaithersburg, Md.), supplemented with bFGF at 40-80 ng/mL, andoptionally containing SCF (15 ng/mL), or Flt3 ligand (75 ng/mL) (see,e.g., Xu et al., (2005) Stem Cells 23(3):315). These media formulationshave the advantage of supporting cell growth at 2-3 times the rate inother systems (see, e.g., WO 03/020920). In some embodiments pPS cellssuch as hES cells may be cultured in a media comprising bFGF and TGFβ.Suitable concentrations of bFGF include about 80 ng/ml. Suitableconcentrations of TGFβ include about 0.5 ng/ml.

In some embodiments, the primate pluripotent stem cells may be platedat >15,000 cells cm⁻² (optimally 90,000 cm⁻² to 170,000 cm⁻²).Typically, enzymatic digestion may be halted before cells becomecompletely dispersed (e.g., about 5 minutes with collagenase IV). Clumpsof about 10 to about 2,000 cells may then be plated directly onto asuitable substrate without further dispersal. Alternatively, the cellsmay be harvested without enzymes before the plate reaches confluence byincubating the cells for about 5 minutes in a solution of 0.5 mM EDTA inPBS or by simply detaching the desired cells from the platemechanically, such as by scraping or isolation with a fine pipette or acell scraper. After washing from the culture vessel, the cells may beplated into a new culture without further dispersal. In a furtherillustration, confluent human embryonic stem cells cultured in theabsence of feeders may be removed from the plates by incubating with asolution of 0.05% (wt/vol) trypsin (Gibco®, Carlsbad, Calif.) and 0.05mM EDTA for 5-15 minutes at 37° C. The remaining cells in the plate maybe removed and the cells may be triturated into a suspension comprisingsingle cells and small clusters, and then plated at densities of50,000-200,000 cells cm⁻² to promote survival and limit differentiation.

In certain embodiments, pPS cells may be cultured on a layer of feedercells, typically fibroblasts derived from embryonic or fetal tissue(Thomson et al. (1998) Science 282:1145). In certain embodiments, thosefeeder cells may be derived from human or murine source. Human feedercells can be isolated from various human tissues or derived bydifferentiation of human embryonic stem cells into fibroblast cells(see, e.g., WO 01/51616) In certain embodiments, human feeder cells thatmay be used include, but are not limited to, placental fibroblasts (see,e.g., Genbacev et al. (2005) Fertil. Steril. 83(5):1517), fallopian tubeepithelial cells (see, e.g., Richards et al. (2002) Nat. Biotechnol.,20:933), foreskin fibroblasts (see, e.g., Amit et al. (2003) Biol.Reprod. 68:2150), uterine endometrial cells (see, e.g., Lee et al.(2005) Biol. Reprod. 72(1):42).

In the practice of the present invention, there are various solidsurfaces that may be used in the culturing of cells. Those solidsurfaces include, but are not limited to, standard commerciallyavailable cell culture plates such as 6-well, 24-well, 96-well, or144-well plates. Other solid surfaces include, but are not limited to,microcarriers and disks. In certain embodiments, the microcarriers maybe used in stirred-tank bioreactors for attachment of the cells. Incertain embodiments, the microcarriers are beads. Those beads come invarious forms such as Cytodex Dextran microcarrier beads with positivecharge groups to augment cell attachment, gelatin/collagen-coated beadsfor cell attachment, and macroporous microcarrier beads with differentporosities for attachment of cells. The Cytodex dextran, gelatin-coatedand the macroporous microcarrier beads are commercially available(Sigma-Aldrich, St. Louis, Mo. or Solohill Engineering Inc., Ann Arbor,Mich.). In certain embodiments, the beads are 90-200 μm in size with anarea of 350-500 cm². Beads may be composed of a variety of materialssuch as, but not limited to, glass or plastic. Disks are sold bycompanies such as New Brunswick Scientific Co, Inc. (Edison, N.J.). Incertain embodiments, the disks are Fibra-cel Disks, which arepolyester/polypropylene disks. A gram of these disks provide a surfacearea of 1200 cm².

The solid surface suitable for growing pPS cells may be made of avariety of substances including, but not limited to, glass or plasticsuch as polystyrene, polyvinylchloride, polycarbonate,polytetrafluorethylene, melinex, or thermanox. In certain embodiments ofthe invention, the solid surfaces may be three-dimensional in shape.Exemplary three-dimensional solid surfaces are described, e.g., in US2005/0031598.

In certain embodiments, the cells are in a single-cell suspension duringthe methods of the invention. The single-cell suspension may beperformed in various ways including, but not limited to, culture in aspinner flask, in a shaker flask, or in a fermentors. Fermentors thatmay be used include, but are not limited to, Celligen Plus (NewBrunswick Scientific Co, Inc., Edison, N.J.), and the STR or theStirred-Tank Reactor (Applikon Inc., Foster City, Calif.). In certainembodiments, the bioreactors may be continuously perfused with media orused in a fed-batch mode. Other suitable bioreactors include the WaveBioreactor bags (GE Healthcare, Piscataway, N.J.). Bioreactors come indifferent sizes including, but not limited to 2.2 liter, 5 liter, 7.5liter, 14 liter or 20 liter, 100 liter, 100 liter, 10,000 liter orlarger.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, developmentalbiology, immunology, neurobiology, endocrinology, cardiology and thelike.

With respect to tissue and cell culture and embryonic stem cells, thereader may wish to refer to any of numerous publications available inthe art, e.g., Teratocarcinomas and Embryonic Stem cells: A PracticalApproach (E. J. Robertson, Ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al., Eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and Uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and R. I. Freshney,Culture of Animal Cells, Wiley-Liss, New York, 2000).

Where derived from an established line of pPS cells, the cellpopulations and isolated cells of this invention can be characterized ashaving the same genome as the line from which they are derived. Thismeans that the chromosomal DNA will be essentially identical by RFLP orby SNP analysis between the pPS cells and the differentiated progenycells (assuming the cells have not been genetically manipulated by thehuman hand). It is contemplated that relatively minute changes in thegenome may occur over time, e.g. in the non-coding regions, howeveroverall the genetic identity will be substantially maintained betweenthe parent cell line and any progeny, e.g. differentiated progeny.Typically the level of genetic identity will be similar to geneticidentity observed between identical twins.

Genetic Alteration of Differentiated Cells

The cells of this invention can be made to contain one or more geneticalterations by genetic engineering of the cells either before or afterdifferentiation (US 2002/0168766 A1). For example in some embodiments,the cells can be processed to increase their replication potential bygenetically altering the cells to express telomerase reversetranscriptase, either before or after they progress to restricteddevelopmental lineage cells or terminally differentiated cells (US2003/0022367 A1).

The cells of this invention can also be genetically altered in order toenhance their ability to be involved in modulating a specifictherapeutic function, or to deliver a therapeutic gene to a site ofadministration. A vector is designed using the known encoding sequencefor the desired gene, operatively linked to a promoter that is eitheractive in all cell types or specifically active in the differentiatedcell type. Alternatively the promoter may be an inducible promoter thatpermits for the timed expression of the genetic alteration. For examplethe cells may be genetically engineered to express a cytokine thatmodulates a cardiac function.

Additional Aspects of the Invention

Additional aspects of the invention include the following:

1. A method of reducing the number of extraneous phenotypic cells in amixed population of cells comprising a) contacting the mixed populationof cells with one or more ligands that specifically bind to theextraneous phenotypic cells; and b) separating the ligand boundextraneous phenotypic cells from the rest of the mixed population ofcells thereby reducing the number of extraneous phenotypic cells from amixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells including theextraneous phenotypic cells.2. The method of 1, wherein the extraneous phenotypic cell is anepithelial cell.3. The method of 2, wherein the epithelial cell expresses a cytokeratin.4. The method of 2, wherein the epithelial cell expresses EpCAM.5. The method of 4, wherein one or more of the cells expressing EpCAMalso expresses TRA-1-60.6. The method of 1, wherein the ligand is an antibody.7. The method of 6, wherein the antibody binds specifically to EpCAM.8. The method of 1, wherein the ligand is bound to a solid support.9. The method of 8, wherein the solid support is a bead.10. The method of 9, wherein the bead is a magnetic bead.11. The method of claim 1, wherein separating the ligand boundextraneous phenotypic cells from the rest of the mixed population ofcells is performed by applying an external force to the mixed populationof cells.12. The method of 11, wherein the external force is provided by amagnetic field.13. The method of 1, wherein the mixed population of cells comprises atargeted phenotype.14. The method of 13, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.15. The method of 14 wherein the targeted phenotype is anoligodendrocyte.16. The method of 14, wherein the targeted phenotype is a cardiomyocyte.17. A method of reducing the number of extraneous phenotypic cells in amixed population of cells comprising a) contacting the mixed populationof cells with one or more ligands that specifically bind to one or moremarkers expressed by an epithelial cell; and b) separating the ligandbound cells of a) from the rest of the mixed population of cells therebyreducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells including the extraneousphenotypic cells.18. The method of 17, wherein the marker expressed by the epithelialcell is a cytokeratin.19. The method of 17, wherein the marker expressed by the epithelialcell is EpCAM.20. The method of 19, wherein the cells expressing EpCAM also expressesTRA-1-60.21. The method of 17, wherein the ligand is an antibody.22. The method of 21, wherein the antibody binds specifically to EpCAM.23. The method of 17, wherein the ligand is bound to a solid support.24. The method of 17, wherein the solid support is a bead.25. The method of 24, wherein the bead is a magnetic bead.26. The method of 17, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.27. The method of 26, wherein the external force is provided by amagnetic field.28. The method of 17, wherein the mixed population of cells comprises atargeted phenotype.29. The method of 28, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.30. The method of 28, wherein the targeted phenotype is anoligodendrocyte.31. The method of 28, wherein the targeted phenotype is a cardiomyocyte.32. A method of reducing the number of extraneous phenotypic cells in amixed population of cells comprising a) contacting the mixed populationof cells with one or more ligands that specifically bind epithelialcells and contacting the mixed population of cells with one or moreligands that specifically bind to undifferentiated pPS cells; and b)separating the ligand bound cells from the rest of the mixed populationof cells thereby reducing the number of extraneous phenotypic cells froma mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells.33. The method of 32, wherein the marker expressed by the epithelialcell is a cytokeratin.34. The method of 32, wherein the marker expressed by the epithelialcell is EpCAM.35. The method of 34, wherein the cells expressing EpCAM also expressTRA-1-60.36. The method of 32, wherein the ligand is an antibody.37. The method of 36, wherein the antibody binds specifically to EpCAM.38. The method of 36, wherein the ligand is bound to a solid support.39. The method of 38, wherein the solid support is a bead.40. The method of 39, wherein the bead is a magnetic bead.41. The method of 32, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.42. The method of 41, wherein the external force is provided by amagnetic field.43. The method of 32, wherein the mixed population of cells comprises atargeted phenotype.44. The method of 43, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.45. The method of 43, wherein the targeted phenotype is anoligodendrocyte.46. The method of 43, wherein the targeted phenotype is a cardiomyocyte.47. The method of 32, wherein the ligand that binds to theundifferentiated pPS cell is an antibody.48. The method of 47, wherein the antibody binds to TRA-1-60.49. The method of 48, wherein the antibody that binds to TRA-1-60 is anIgG.50. The method of 48, wherein the antibody that binds to TRA-1-60 is anIgM51. The method of 32, wherein the ligand that binds to an extraneousphenotypic cell is an antibody that specifically binds to EpCAM and theligand that binds to an undifferentiated pPS cell is an antibody thatbinds to TRA-1-60.52. The method of 51, wherein the antibody that binds to EpCAM is linkedto a magnetic bead and the antibody that binds to TRA-1-60 is linked toa magnetic bead.53. A method of reducing the number of extraneous phenotypic cells in amixed population of cells comprising a) contacting the mixed populationof cells with one or more ligands that specifically bind to EpCAM and b)separating the EpCAM bound cells from the rest of the mixed populationof cells thereby reducing the number of extraneous phenotypic cells froma mixed population of cells, wherein the mixed population of cellscomprises the in vitro differentiated progeny of pPS cells.54. The method of 53, wherein the ligand that binds EpCAM is anantibody.55. The method of 54, wherein the EpCAM antibody is linked to a solidsupport.56. The method of 55, wherein the solid support is a bead.57. The method of 56, wherein the bead is a magnetic bead.58. The method of 53, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.59. The method of 58, wherein the external force is provided by amagnetic field.60. The method of 53, wherein the mixed population of cells comprises atargeted phenotype.61. The method of 60, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.62. The method of 43, wherein the targeted phenotype is anoligodendrocyte.63. The method of 43, wherein the targeted phenotype is a cardiomyocyte.64. A method of reducing the number of extraneous phenotypic cells in amixed population of cells comprising a) contacting the mixed populationof cells with one or more ligands that specifically bind to EpCAM andone or more ligands that specifically bind to TRA-1-60; and b)separating the cells bound to the ligand for EpCAM and the cells boundto the ligand of TRA-1-60 from the rest of the mixed population of cellsthereby reducing the number of extraneous phenotypic cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells.65. The method of 64, wherein the ligand that binds EpCAM is anantibody.66. The method of 64, wherein the ligand that binds EpCAM is linked to asolid support.67. The method of 66, wherein the solid support is a bead.68. The method of 67, wherein the bead is a magnetic bead.69. The method of 64, wherein the ligand that binds TRA-1-60 is anantibody.70. The method of 69, wherein the antibody is an IgG.71. The method of 69, wherein the antibody is an IgM.72. The method of 64, wherein the ligand that binds TRA-1-60 is linkedto a solid support.73. The method of 72, wherein the solid support is a bead.74. The method of 73, wherein the bead is a magnetic bead.75. The method of 64, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.76. The method of 75, wherein the external force is provided by amagnetic field.77. The method of 64, wherein the mixed population of cells comprises atargeted phenotype.78. The method of 77, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.79. The method of 77, wherein the targeted phenotype is anoligodendrocyte.80. The method of 77, wherein the targeted phenotype is a cardiomyocyte.81. A method of reducing the number of clustered epithelial formingcells in a mixed population of cells comprising a) contacting the mixedpopulation of cells with one or more ligands that specifically bind toEpCAM and b) separating the EpCAM bound cells from the rest of the mixedpopulation of cells thereby reducing the number of clustered epithelialforming cells from a mixed population of cells, wherein the mixedpopulation of cells comprises the in vitro differentiated progeny of pPScells.82. The method of 81, wherein the ligand that binds EpCAM is anantibody.83. The method of 82, wherein the EpCAM antibody is linked to a solidsupport.84. The method of 83, wherein the solid support is a bead.85. The method of 84, wherein the bead is a magnetic bead.86. The method of 81, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.87. The method of 81, wherein the external force is provided by amagnetic field.88. The method of 88, wherein the mixed population of cells comprises atargeted phenotype.89. The method of 88, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.90. The method of 88, wherein the targeted phenotype is anoligodendrocyte.91. The method of 88, wherein the targeted phenotype is a cardiomyocyte.92. A method of reducing the number of cells expressing one or moremolecules expressed by undifferentiated cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically an epithelial cell; and b) separating theligand bound cells of a) from the rest of the mixed population of cellsthereby reducing the number of undifferentiated cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells.93. The method of 92, wherein the ligand is an antibody.94. The method of 92, wherein the antibody specifically binds to EpCAM.95. The method of 92, wherein the antibody specifically binds to acytokeratin.96. The method of 92, wherein the antibody is linked to a solid support97. The method of 96, wherein the solid support is a bead.98. The method of 97, wherein the bead is a magnetic bead.99. The method of 92, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.100. The method of 99, wherein the external force is provided by amagnetic field.101. The method of 92, wherein the mixed population of cells comprises atargeted phenotype.102. The method of 101, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.103. The method of 101, wherein the targeted phenotype is anoligodendrocyte.104. The method of 101, wherein the targeted phenotype is acardiomyocyte.105. The method of 92 wherein the molecule expressed by anundifferentiated cell is TRA-1-60.106. A method of reducing the number of cells expressing one or moremolecules expressed by undifferentiated cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to EpCAM and b) separating theligand bound EpCAM cells from the rest of the mixed population of cellsthereby reducing the number of undifferentiated cells from a mixedpopulation of cells, wherein the mixed population of cells comprises thein vitro differentiated progeny of pPS cells.107. The method of 106, wherein the molecule expressed by theundifferentiated cell is TRA-1-60.108. The method of 106, wherein the ligand that binds EpCAM is anantibody.109. The method of 108, wherein the EpCAM antibody is linked to a solidsupport.110. The method of 108, wherein the solid support is a bead.111. The method of 110, wherein the bead is a magnetic bead.112. The method of 106, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.113. The method of 112, wherein the external force is provided by amagnetic field.114. The method of 106, wherein the mixed population of cells comprisesa targeted phenotype.115. The method of 114, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.116. The method of 114, wherein the targeted phenotype is anoligodendrocyte.117. The method of 114, wherein the targeted phenotype is acardiomyocyte.118. A method of reducing the number of cells expressing one or moremolecules expressed by undifferentiated cells in a mixed population ofcells comprising a) contacting the mixed population of cells with one ormore ligands that specifically bind to EpCAM and one or more ligandsthat specifically bind to TRA-1-60 and b) separating the ligand boundcells from the rest of the mixed population of cells thereby reducingthe number of undifferentiated cells from a mixed population of cells,wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.119. The method of 118, wherein the ligand that binds EpCAM is anantibody.120. The method of 119, wherein the ligand that binds EpCAM is linked toa solid support.121. The method of 120, wherein the solid support is a bead.122. The method of 121, wherein the bead is a magnetic bead.123. The method of 118, wherein the ligand that binds TRA-1-60 is anantibody.124. The method of 118, wherein the antibody is an IgG.125. The method of 118, wherein the antibody is an IgM.126. The method of 118, wherein the ligand that binds TRA-1-60 is linkedto a solid support.127. The method of 126, wherein the solid support is a bead.128. The method of 127, wherein the bead is a magnetic bead.129. The method of 118, wherein separating the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.130. The method of 129, wherein the external force is provided by amagnetic field.131. The method of 118, wherein the mixed population of cells comprisesa targeted phenotype.132. The method of 131, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.133. The method of 131, wherein the targeted phenotype is anoligodendrocyte.134. The method of 131, wherein the targeted phenotype is acardiomyocyte.135. A method of obtaining a population of cells comprising the in vitrodifferentiated progeny of pPS cells that is essentially free ofextraneous phenotypic cells comprising a) obtaining a population ofcells comprising the in vitro differentiated progeny of pPS cells; b)contacting the cell population of a) with one or more ligands that bindto epithelial cells and c) removing the ligand bound cells of b) therebyobtaining a population of cells that is essentially free of extraneousphenotypic cells.136. The method of 135, wherein the ligand that binds the epithelialcell is EpCAM.137. The method of 135, wherein the ligand that binds EpCAM is anantibody.138. The method of 135, wherein the ligand that binds EpCAM is linked toa solid support.139. The method of 138, wherein the solid support is a bead.140. The method of 139, wherein the bead is a magnetic bead.141. The method of 135, wherein removing the ligand bound extraneousphenotypic cells from the rest of the mixed population of cells isperformed by applying an external force to the mixed population ofcells.142. The method of 141, wherein the external force is provided by amagnetic field.143. The method of 135, wherein the mixed population of cells comprisesa targeted phenotype.144. The method of 143, wherein the targeted phenotype is chosen from anoligodendrocyte, a cardiomyocyte, an islet cell, a hematopoietic cell, ahepatocyte, an osteoblast and a chondrocyte.145. The method of 143, wherein the targeted phenotype is anoligodendrocyte.145. The method of 143, wherein the targeted phenotype is acardiomyocyte.146. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing a molecule foundon an epithelial cell.147. The mixed population of cells of 146, wherein the at least one cellexpressing a molecule found on an epithelial cell expresses EpCAM.148. The mixed population of cells of 143, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoietic cell, a hepatocyte, an osteoblast and achondrocyte.149. The mixed population of cells of 146, wherein the targetedphenotype is an oligodendrocyte.150. The mixed population of cells of 146, wherein the targetedphenotype is a cardiomyocyte.151. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing a molecule foundon an epithelial cell and at least one cell expressing a molecule foundon an undifferentiated pluripotent stem cell.152. The mixed population of cells of 151, wherein the at least one cellexpressing a molecule found on an epithelial cell expresses EpCAM.153. The mixed population of cells of 151, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoietic cell, a hepatocyte, an osteoblast and achondrocyte.154. The mixed population of cells of 151, wherein the targetedphenotype is an oligodendrocyte.155. The mixed population of cells of 151, wherein the targetedphenotype is a cardiomyocyte.156. The mixed population of cells of 151, wherein the at least one cellexpressing a molecule found on an undifferentiated cell expressesTRA-1-60.157. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing EpCAM.158. The mixed population of cells of 157, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoietic cell, a hepatocyte, an osteoblast and achondrocyte.159. The mixed population of cells of 157, wherein the targetedphenotype is an oligodendrocyte.160. The mixed population of cells of 157, wherein the targetedphenotype is a cardiomyocyte.161. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing a cytokeratin.162. The mixed population of cells of 161, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoietic cell, a hepatocyte, an osteoblast and achondrocyte.163. The mixed population of cells of 161, wherein the targetedphenotype is an oligodendrocyte.164. The mixed population of cells of 161, wherein the targetedphenotype is a cardiomyocyte.165. The mixed population of cells of 161, wherein the the at least onecell expressing a cytokeratin also expresses EpCAM.166. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing a cytokeratinand at least one cell expressing a molecule found on an undifferentiatedcell.165. The mixed population of cells of 166, wherein the at least one cellexpressing a cytokeratin also expresses EpCAM.167. The mixed population of cells of 166, wherein the at least one cellexpressing a molecule found on an undifferentiated cell expressesTRA-1-60.168. The mixed population of cells of 166, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoietic cell, a hepatocyte, an osteoblast and achondrocyte.

169. The mixed population of cells of 166, wherein the targetedphenotype is an oligodendrocyte.

170. The mixed population of cells of claim 166, wherein the targetedphenotype is a cardiomyocyte.

171. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing EpCAM and atleast one cell expressing TRA-1-60.

172. The mixed population of cells of 171, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoictic cell, a hepatocyte, an osteoblast and achondrocyte.

173. The mixed population of cells of 171, wherein the targetedphenotype is an oligodendrocyte.

174. The mixed population of cells of 171, wherein the targetedphenotype is a cardiomyocyte.

175. A mixed population of cells that is enriched for a targetedphenotype, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells and wherein the mixed population ofcells has been depleted of at least one cell expressing a cytokeratinand at least one cell expressing TRA-1-60.

176. The mixed population of cells of 175, wherein the at least one cellexpressing a cytokeratin also expresses EpCAM.

177. The mixed population of cells of 175, wherein the targetedphenotype is chosen from an oligodendrocyte, a cardiomyocyte, an isletcell, a hematopoietic cell, a hepatocyte, an osteoblast and achondrocyte.

178. The mixed population of cells of 175, wherein the targetedphenotype is an oligodendrocyte.

179. The mixed population of cells of 175, wherein the targetedphenotype is a cardiomyocyte.

181. A kit for depleting cells having an extraneous phenotype from amixed population of cells comprising a) a ligand for one or moremolecules expressed on an epithelial cell; b) a ligand for one or moremolecules expressed on an undifferentiated cell; and c) one or morecontainers, wherein the mixed population of cells comprises the in vitrodifferentiated progeny of pPS cells.

182. A kit for depleting cells having an extraneous phenotype from amixed population of cells comprising a ligand for EpCAM, a ligand forTRA-1-60 and one or more containers, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cells.

In the following Examples all experiments utilizing human embryoniccells (hES) cells were performed using established publicly availablehES cell lines.

EXAMPLES Example 1 Epithelial Cell Markers Correlate with ExtraneousPhenotypes in Oligodendrocytes Differentiated In Vitro from PrimatePluripotent Stem Cells

Total RNA was extracted, according to the manufacturer's instructionsusing a Qiagen RNAEasy Kit (Qiagen, Valencia Calif.) fromoligodendroglial progenitor cells differentiated in vitro from humanembryonic stem cells. Vials containing 7.5×10⁶ oligodendroglialprogenitor cells each were used. These vials originated from three lotsthat generated a high number of clustered epithelial structures (CES) invivo, and three lots that had low levels of CES in vivo. RNA integritywas confirmed using the Agilent bioanalyzer, (Agilent, Santa Clara,Calif.) and all samples used had an RNA integrity number (RIN) above9.8. The 3′ IVT Express kit (Affymetrix, Santa Clara, Calif.) was usedaccording to the manufacturer's instructions to generate cDNA from thisRNA. Microarray analysis was performed according to the manufacturer'sinstructions with these cDNA samples using the Affymetrix U133+2.0genome array (Affymetrix, Santa Clara, Calif.). This array is acomprehensive human genome expression array containing more than 54,000probe sets. Genes with greater than a 2-fold difference in expressionbetween these groups of high and low CES lots were examined further.Table 1 shows that increased expression of epithelial cell markerscorrelates with increased expression of CES in lots of GRNOPC1.

TABLE 1 Fold expression difference (3 High CES Gene ID Full Name vs. 3Low CES lots) KRT19 Keratin 19 3.3 EPPK1 Epiplakin 1 3.1 2.8 DSG2Desmoglein 2 3.1 2.9 DSC3 Desmocollin 3 2.6 2.5 EpCAM Epithelial celladhesion marker 2.3

Example 2 Identification of Epithelial Structures in Rat Spinal Cords

Rats that were administered human oligodendroglial progenitor cellswhich were differentiated in vitro from hES cells on rare occasionsdeveloped clustered epithelial structures. FIG. 1 shows clusteredepithelial structures stained with hematoxylin and eosin. The structureswere identified as epithelial cells based on morphology.

To further characterize these clustered epithelial structuresimmunohistochemistry (IHC) was performed using antibodies topan-cytokeratin or an antibody to EpCAM. The pan-cytokeratin antibodies(Millipore, Billerica, Mass.) were a mixture of two antibodies: anti-AE1and anti-AE3. The AE1 antibody recognizes keratins CK10, CK14, CK15, andCK16 as well keratin CK19. The AE3 antibody recognizes keratins CK1,CK2, CK3, CK4, CK5 and CK6, as well as keratins CK7 and CK8. KeratinCK10 is expressed in squamous keratinzed skin and some gut cells.Keratin CK19 is expressed in secretory epithelia, most glands,pseudostratified epithelia, secretory gut and respiratory epithelia.Keratin CK8 typically co-expresses with keratin CK18 and is found inmost glands, secretory gut, and most secretory epithelia.

The EpCAM antibody used was Ber-Ep4 (Invitrogen, Carlsbad, Calif.).EpCAM is expressed at the basolateral membrane of most epithelialtissues and epithelial lineage cells. It is not expressed in squamousepithelium. It may be transiently expressed in association with cellularproliferation.

Sections were examined using (IHC) with pan-cytokeratin antiserum tolabel epithelial cells. Slides were deparaffinized using xylenes and adecreasing sequential ethanol series. Antigen retrieval pretreatmentconsisted of incubating the slides with 4% pepsin in DPBS for 30 minutesat 37° C., followed by cooling to ambient temperature. Slides wererinsed in DPBS and incubated with blocking buffer (0.3% Triton X-100,10% normal goat serum, 1% bovine serum albumin, 3% H₂O₂ in DPBS) for 1hour at ambient temperature followed by incubation with primaryantiserum raised against pan-cytokeratin diluted 1:400, (catalogue #mAB3412 clone AE1/AE3) (Millipore, Billerica, Mass.) for 24 hours atambient temperature. The sections were washed and incubated withbiotinylated secondary antibody diluted 1:500, (catalogue #BA-1000)(Vector Laboratories, Burlingame, Calif.) for 1 hour at ambienttemperature, washed and incubated with streptavidin-HRP diluted 1:1000in DPBS (Vectastain Elite ABC kit, Vector Laboratories) for 1 hour atambient temperature. Ni-DAB, (Vector Laboratories) was used for HRPvisualization. Slides were dehydrated through an increasing sequentialethanol series, defatted in xylenes and coverslipped with Permount™(Fisher Scientific, Hampton, N.H.). The slides were examined under alight microscope.

Sections were examined using IHC with EpCAM antiserum to labelepithelial cells. Slides were deparaffinized using xylenes and adecreasing sequential ethanol series. Antigen retrieval pretreatmentconsisted of incubating the slides with 4% pepsin in DPBS for 30 minutesat 37° C., followed by cooling to ambient temperature. Slides wererinsed in DPBS and incubated with blocking buffer (0.3% Triton X-100,10% normal goat serum, 1% bovine serum albumin, 3% H₂O₂ in DPBS) for 1hour at ambient temperature followed by incubation with primaryantiserum raised against EpCAM diluted 1:100, (Ber-EP4; catalogue #mAB84clone; Dako; Carpinteria, Calif.) for 24 hours at ambient temperature.The sections were washed and incubated with biotinylated secondaryantibody diluted 1:500, (catalogue #BA-1000) (Vector Laboratories,Burlingame, Calif.) for 1 hour at ambient temperature, washed andincubated with streptavidin-HRP diluted 1:1000 in DPBS (Vectastain EliteABC kit, Vector Laboratories) for 1 hour at ambient temperature. Ni-DAB,(Vector Laboratories) was used for HRP visualization. Slides weredehydrated through an increasing sequential ethanol series, defatted inxylenes and coverslipped with Permount™ (Fisher Scientific, Hampton,N.H.). The slides were examined under a light microscope.

FIG. 2 demonstrates that human tissue array (containing liver, smallintestine, spleen, stomach, colon and skin cells) all labeled forpan-cytokeratin. The right side of FIG. 2 demonstrates that murineintramuscular teratomas (in SKID/bg mice) also labeled forpan-cytokeratin. FIG. 3 shows similar results for the EpCAM antibody.

FIG. 4 shows that the clustered epithelial structures in 3 distinctanimals labeled positively for pan-cytokeratin. Magnifications shown are24× (upper panels) and 400× (lower panels). FIG. 5 shows the clusteredepithelial structure in one animal labeled with the EpCAM antibody.Magnifications shown are 24× (upper left panel) and 400× (lower panels).FIG. 6 shows the clustered epithelial structures in one animal labeledfor both pan-cytokeratin and EpCam. Magnifications shown are 24× (upperpanels) and 400× (lower panels). FIG. 7 shows that the clusteredepithelial structures in one animal labeled for pan-cytokeratin, but notfor EpCAM. Magnifications shown are 24× (upper panels) and 400× (lowerpanels). FIG. 8 shows that clustered epithelial structures in one animaldid not label for either pan-cytokeratin or EpCAM. Magnifications shownare 24× (upper panels) and 400× (lower panels).

Example 3 Characterization of Mixed Populations of Cells ComprisingOligodendrocytes

hES cells were differentiated into oligodendrocytes and oligodendrocyteprecursors as described above and in U.S. Pat. No. 7,285,415. Twoseparate lots (M07D1A and 2008-05A-ms) were generated. The cells in bothpreparations were characterized by flow cytometry by labeling the cellswith antibodies for the following epithelial markers: EpCAM (cloneBER-Ep4) conjugated to ALexa fluor 647 (Invitrogen, Carlsbad, Calif.)and a marker associated with primate undifferentiated pluripotent stemcells: TRA-1-60 (Invitrogen, Carlsbad, Calif.). The secondary antibodyused for TRA-1-60 was an anti mouse IgM A488 conjugated with AlexaFluor® (Invitrogen, Carlsbad, Calif.).

Briefly, 5×10⁵ cells were labeled per reaction in a volume of 100 μL.For primary antibodies 500 ng per label were used. The cells wereincubated on ice for 15 minutes and then washed two times with stainingbuffer (0.5% Human serum albumin PBS). Secondary antibodies were diluted1:400 and labeled on ice for 15 minutes. The cells were washed two timeswith staining buffer and then resuspended in propidium iodine stainingbuffer (prepared by adding 10 μL of a 1.0 mg/ml stock of propidiumiodine to 2 ml of staining buffer for a final concentration of 5 μg/mLof propidium iodine). The cells were filtered using a FACS tube with acell strainer (BD Bioscience, San Jose, Calif.). Cells were gated forpropidium iodine vs. forward scatter and then again for forward scattervs. side scatter. Within these gates 20,000 events were collected. Theresults are shown in FIG. 9 and indicate a small percentage of thepopulation expressed pan-cytokeratin (both lots) and EpCAM (leftpanels). Most of the cells expressing EpCAM also labeled forpan-cytokeratin. Double positive cells are clearly evident.

Example 4 EpCAM Antibodies Successfully Remove EpCAM+ Cells from a MixedPopulation of Cells

In this experiment, an EpCAM negative cell line (Human Embryonic Kidney293 cells) was spiked with EpCAM positive cell line (humanadenocarcinoma line SW480) to create a mixed population that was 10%EpCAM+. The cells were labeled with the BER-Ep4 EpCAM antibody(Invitrogen, Carlsbad, Calif.) conjugated to a magnetic Dynal bead(CELLection Epithelial Enrich Bead) (Invitrogen, Carlsbad, Calif.) andthe cells were separated using the Dynal CELLection kit (Invitrogen,Carlsbad, Calif.) according to the protocol below.

The Dynabeads (Invitrogen, Carlsbad, Calif.) were resuspended in thevial. 50 μL/2e7 cells of Dynabeads were transferred to a 15 mL tube. Thesame volume of Buffer 1 (PBS/0.1% Human Serum Albumin), or at least 1 mLwas added to the tube and mixed. The tube was placed in the magnet for 1minute and the supernatant was discarded. The tube was removed from themagnet and the washed. Dynabeads were resuspended in the same volume ofBuffer 1 as the initial volume of Dynabeads.

To prepare the sample 2e7/mL cells were resuspended in cold Buffer 2(PBS/0.1% Human Serum Albumin/2 mM EDTA). To enrich for EpCAM+ cells 50μL of Dynabeads was added to 1 mL of prepared sample and then incubatedfor 30 minutes at 2-8° C. in MACSmix, (Invitrogen, Carlsbad, Calif.) setat gentle rotation. The tube was placed in the magnet for 2 minutes. Thesupernatant containing the EpCAM-depleted cells was reserved on ice.

The EpCAM+ bead-bound cells were washed 3× in preheated (37° C.) Buffer3 (RPMI/0.1% HSA/1 mM CaCL₂/4 mM MgCl₂) (5 mL per wash) and placed inthe magnet. All washes were pooled in a single tube. The cells wereresuspended in 200 μL (per 2e7 cells) preheated (37° C.) Buffer 3. Torelease the EpCAM+ cells from the beads 4 uL of Releasing Buffer (DNAse)(provided in the kit) (Invitrogen, Carlsbad, Calif.) was added to thetube. The tube was incubated for 15 minutes at room temperature in aMACSmix, (Invitrogen, Carlsbad Calif.) set at gentle rotation. Thecontents of the tube were pipetted vigorously with a 1 mL pipette atleast 5-10 times and then placed in the magnet for 2 minutes. Thesupernatant with released cells was transferred into a 15 mL tube precoated with RPMI media. Samples were pooled. The beads were resuspendedin Buffer 3 (200 μL) and the beads were once again pipetted vigorouslyand returned to the magnet for 2 minutes. The supernatant contained theunbound EpCAM+ cells.

The cell fractions were labeled for EpCAM using the protocol describedin Example 3. An isotype control (murine IgG1) (eBioscience, San Diego,Calif.) for the EpCAM antibody was used. The results are shown in FIG.10. The data demonstrated that EpCAM+ cells could be successfullydepleted from a mixed population of cells (see FIG. 9 bottom 3^(rd) and4^(th) panel). Efficiency of recovery of the EpCAM negative cells wascalculated to be 93%. Efficiency of recovery of the EpCAM positive cellswas calculated to be 9%.

Example 5 Depleting EpCAM+ Cells Simultaneously DepletesPan-Cytokeratin+ Cells and TRA-1-60 Cells

In this experiment, anti-EpCAM Dynabeads (Invitrogen, Carlsbad, Calif.)were resuspended in the vial and 25 beads per target EpCAM expressingcell were transferred to a 15 mL tube (assumed to be 5% of the targetpopulation—5% was chosen as an estimate of the number of EpCAM+ cells inthe population. 5% is higher than the upper limit seen in previouspopulations of oligodendroglial progenitors differentiated in vitro fromhES cells). The same volume of Buffer 1 (PBS/0.1% Human Serum Albumin),or at least 1 mL was added to the tube and mixed in order to wash thebeads prior to use. The tube was placed in the magnet for 1 minute andthe supernatant was discarded. The tube was removed from the magnet andthe washed. Dynabeads were resuspended in the same volume of Buffer 1 asthe initial volume of Dynabeads.

To prepare the sample 2e7/mL cells (mixed population of cells comprisingoligodendroglial progenitor cells differentiated in vitro from hEScells) were resuspended in cold Buffer 2 (PBS/0.1% Human Serum Albumin/2mM EDTA). To deplete EpCAM+ cells the Dynabeads were added to theprepared sample and then incubated for 30 minutes at 2-8° C. in MACSmix,(Invitrogen, Carlsbad, Calif.) set at gentle rotation. The tube wasplaced in the magnet for 2 minutes. The supernatant containing theEpCAM-depleted cells was reserved on ice.

The cell fractions were labeled for EpCAM, TRA-1-60 and PCK using theprotocol described in Example 3. Isotype controls (murine IgG1, murineIgM) (eBioscience, San Diego, Calif.) for the antibodies were used. Theresults are shown in FIG. 11. The data demonstrated that EpCAM+ cellscould be successfully depleted from a mixed population of cells (seeFIG. 11 bottom 3rd and 4th panel). Efficiency of recovery of the EpCAMnegative cells was calculated to be 96%. In addition, EpCAM depletionreduced the percentages of both the TRA-1-60+ and PCK+ cell populations.

Example 6 EpCAM/TRA 1-60 Depleted Human Oligodendroglial ProgenitorCells Formed Fewer Epithelial Structures in Rat Spinal Cords

This experiment investigated the effects of depletion of extraneousphenotypes on clustered epithelial formation in vivo using the rat as ananimal model. Lots of human oligodendroglial progenitors expected to behigh in clustered epithelial structures were chosen specifically forthis study. Two groups of rats with spinal cord contusion injuries atthe thoracic level received intraspinal cord injections of humanoligodendroglial progenitor cells which either had, or had not been,depleted of EpCAM+/TRA 1-60+ cells. Six months later, animals wereperfused and spinal cords were removed and sectioned on the longitudinalplane. The spinal cord tissue extending approximately 1 cm rostral and 1cm caudal to the area of injury was stained with hematoxylin and eosinand examined for clustered epithelial structures. Results are shown inTable 2.

TABLE 2 Frequency of Epithelial Structure Formation Following Depletionof EpCAM+/TRA 1-60+ Cells Number of Percent of Animals Animals withEpithelial with Epithelial Cell Type Structures StructuresOligodendroglial Progenitor Cells 6:10 60% Differentiated in vitro fromhES Cells Oligodendroglial Progenitor Cells 8:23 35% Differentiated invitro from hES Cells Depleted of EpCAM+ Cells

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

1-18. (canceled)
 19. A method of reducing the number of extraneousphenotypic cells in a mixed population of cells comprisingoligodendrocyte progenitor cells that are the in vitro differentiatedprogeny of pPS cells and extraneous phenotypic cells expressing Cd49f,the method comprising: a) contacting the mixed population of cells withone or more ligands that specifically bind to Cd49f; and b) separatingthe ligand-bound Cd49f expressing cells from the rest of the mixedpopulation of cells, thereby reducing the number of extraneousphenotypic cells in the mixed population of cells.
 20. The method ofclaim 19, wherein the ligand is an antibody.
 21. The method of claim 19,wherein the ligand is bound to a solid support.
 22. The method of claim21, wherein the solid support is a bead.
 23. The method of claim 22,wherein the bead is a magnetic bead.
 24. The method of claim 19, whereinthe extraneous phenotypic cells are epithelial lineage cells.
 25. Themethod of claim 19, further comprising contacting the mixed populationof cells with one or more ligands that specifically bind to EpCAM beforestep b).
 26. The method of claim 19, further comprising contacting themixed population of cells with one or more ligands that specificallybind to EpCAM after step b).
 27. A mixed population of cells enrichedfor oligodendrocyte progenitor cells, wherein the mixed population ofcells comprises the in vitro differentiated progeny of pPS cells andwherein the mixed population has a reduced number of extraneousphenotypic cells expressing Cd49f.
 28. The mixed population of cells ofclaim 27, wherein the extraneous phenotypic cells are epithelial lineagecells.
 29. The mixed population of cells of claim 27, wherein the mixedpopulation also has a reduced number of extraneous phenotypic cellsexpressing EpCAM.
 30. The mixed population of cells of claim 27, whereinthe mixed population also has a reduced number of extraneous phenotypiccells expressing a cytokeratin.
 31. A container comprising a compositioncomprising a population of oligodendrocyte progenitor cells depleted ofextraneous phenotypic cells expressing Cd49f, wherein theoligodendrocyte progenitor cells and the extraneous phenotypic cells arethe in vitro differentiated progeny of pPS cells.
 32. The containeraccording to claim 31, wherein the extraneous phenotypic cells areepithelial lineage cells.
 33. The container according to claim 31,wherein the extraneous phenotypic cells further express EpCAM.
 34. Thecontainer according to claim 31, wherein the extraneous phenotypic cellsfurther express a cytokeratin.